Enclosed filtration system processes

ABSTRACT

A product concentration device that utilizes a reservoir connected to a hollow-fiber filter element where the reservoir can serve as a container for filtrate emanating from another filtering device, such that product in the reservoir can be stored, concentrated and/or further processed as desired. Enclosed reactor systems, each of at least three chambers, fluid flow between the chambers controlled by selectively permeable barriers, flow controlled by an alternating flow diaphragm pump.

CROSS REFERENCE TO OTHER APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/269,409, filed on Sep. 19, 2016, which is a continuation of U.S.application Ser. No. 13/408,243, filed on Feb. 29, 2012, now U.S. Pat.No. 9,446,354, which is a Continuation-In-Part (CIP) of PCT ApplicationNo. PCT/US2011/001485, filed on Aug. 24, 2011, which claims the benefitof U.S. provisional application Ser. No. 61/376,810, filed on Aug. 25,2010. The contents of the above applications are incorporated herein byreference in their entireties.

FIELD OF INVENTION

The present invention relates to filtration systems. More specifically,the invention relates to filtration systems for biological fluids andproducts, as well as sampling manifolds, pump systems, and modifyingmodules useful in such systems.

BACKGROUND

Filtration is typically performed to separate, clarify, modify and/orconcentrate a fluid solution, mixture or suspension. In thebiotechnology pharmaceutical and medical industries, filtration is vitalfor the successful production, processing and analysis of drugs,diagnostics, chemicals as well as many other products. As examples,filtration may be used to sterilize fluids or gases, clarify a complexsuspension into a filtered “clear” fraction and an unfiltered fraction;similarly, constituents in a suspension may be concentrated by removingor “filtering out” the suspending medium. Further, with appropriateselection of filter material, filter pore size or other filtervariables, many other specialized filter uses have been developed; thesemay involve selective isolation of constituents from various sources,including, cultures of microorganisms, blood, as well as other fluidsthat may be solutions mixtures or suspensions. With further advancementsin cell and recombinant DNA technologies many new products are beingdeveloped, many of which are so complex that they can only be producedby the complex synthetic machinery of live cells, using cell culturetechniques. Filtration may be used to enhance the productivity of suchcell cultures ‘I’ by maintaining the cultures for extended periods athigh cell concentrations at high productivity and by providing a productstream more amenable to further processing and purification.

Filter chemistries, configurations and modalities of use have beendeveloped to facilitate separation of materials according to theirchemical and physical properties; In spite the extensive developments infilter technology, they are generally limited by their tendency to clog;for example, when used to filter a suspension of cultured mammaliancells they tend to clog with dead cells, cell debris, aggregates,fibrous biomolecules or other constituents found in the complex “soup”of a culture. In this regard, the method of filtration can have aprofound effect on the filtration efficiency and the longevity of themembrane. In one kind of filtration process, commonly known as “deadend” filtration, the entire fluid is passed through the membraneperpendicular to the membrane surface. Debris rapidly accumulates at thesurface resulting in rapid blockage of the membrane. Typically,application using dead end filtration involves small samples. Theprocess is simple and relatively inexpensive. Another filtrationprocess, generally known as Tangential Flow Filtration (also known asTFF) offers an improvement over dead end filtration. In TFF, fluid to befiltered is recirculated with a pump, typically, from a reservoirthrough a filter and back to the reservoir. The flow through the filteris parallel to the surface of the filter. Any accumulation of debris iseffectively removed by the “washout” effect of the circulating fluid;nevertheless, one of its limitations is the tendency to form agelatinous deposit on the filter surface, which may limit theeffectiveness of the filter and eventually clogging it. Anotherprocesses, known as alternating tangential flow filtration, offers yetanother mode of filtration; It is similar to TFF, in that it generates aflow pattern parallel to the filtration membrane surface; however, itdiffers from TFF in that the direction of flow is repeatedly alternatingor reversing across the filter surface. If a change in flow direction isdescribed using a complex pathway of tubing, valves and pumps, placementof such components in a culture flow path adds sheer to the system andprovides sites for cell to aggregate and potential clogging sites, norare such systems very amenable for sustaining homogeneous cultures. Thealternating tangential flow filtration system described in U.S. Pat. No.6,544,424 consists of a filter element, commonly a hollow fibercartridge, connected at one end to a reservoir containing the content tobe filtered and at the other end the filter its connected to a diaphragmpump capable of receiving and reversibly expelling the unfiltered liquidflowing reversibly between reservoir and pump through the filterelement. The system has shown the ability to sustain filtration of acomplex mixtures, Including the medium of a cell culture, even when thatmedium is burdened with high cell concentration and other cellularproducts. That system, however, is limited in its range of applications.

The use of animal cell culture is increasingly used for production ofvarious cell derived biologicals that may be natural or engineered,including proteins, nucleotides, metabolites and many others.Accordingly, methods of production may also vary. They may range fromthe use of “simple” batch to continuous processes. In a normal batchculture production processes, cells are first inoculated into a freshmedium, after which the cells rapidly enter a logarithmic growth phase.As they consume the nutrients in the medium, waste products accumulate;concomitantly, cells transition from rapid growth to a stationary growthphase followed by a cellular decay phase. While several methods havebeen developed to optimize batch culture production, in each case, theseprocesses undergo rapid growth and decay cycles. Another culture processinvolves maintenance of the culture continuously using a processcommonly known as perfusion. In a perfusion culture, waste productsgenerated by the cells are continuously removed from the culture, whileretaining the cells. Removed waste medium is replenished with freshmedium. With this method, it is possible, therefore, to achieve a stateof equilibrium in which cell concentration and productivity aremaintained. Typically, about one to two culture volumes are exchangedper day and the cell concentration achieved in perfusion is typically 2to more than 10 times that achieved at the peak of batch culture. Yet,in spite of the great benefits of the perfusion process, its acceptancehas been slow. One reason for this slow acceptance may be inherent inthe fact that most products originate at small scale in a batch culturesystem, like a “T” flask. If more material is needed, it is generallyproduced by increasing the number and size of the “T” flasks ortransferring the culture to roller bottles or spinner flasks, both ofwhich are typically also batch cultures by nature. By the time theculture is scaled to a bioreactor, the process has been largely biasedby the previous handling of the culture. It would therefore be desirableto create a disposable perfusion system that is more accessible at smallscale, at the level of research and development. Attempts to addressthis issue with hollow fiber bioreactor or other solid bed bioreactor,in which the cells grow attached or entrapped to a fixed surface, areonly partially effective; their inherent inhomogeneity andinaccessibility to the cells limits their usefulness as a research tool.It would desirable to create a system that is easily scaled down, thatwould maintain cultures homogeneously in continuous perfusion, such thatsampling, modifying or monitoring any part of the culture will reflectthe conditions in the entire culture. An investigator may tap into sucha continuous culture as the need arises for cells, for analysis or for adesired product to study the behavior of the cells in a continuoussteady state culture. An investigator can make essential modificationsto such a culture followed by observing the cultures response. Inaddition to proving a means for generating product, such a continuousculture may offer a powerful research and development tool. The proposedinvention addresses this issue by providing a perfusion bioreactorsystem, that maintains a homogenous culture which is accessible tomanipulation, sampling and analysis. The proposed system may be providedin a convenient sterile form ready for use and readily disposable.

With advancements in new materials, manufacturing methods andrequirements in recent years, the construction and use of disposableequipment has gained increasing acceptance. The use of disposable bagsas cell culture bioreactors and storage vessels has become more common.Such disposable containers can be “setup” with minimal handling and donot require cleaning or sterilization by the user. They are suppliedclean, sterile and in a form ready for use, at great savings in cost andreduced handling by the user; furthermore, at the end of their use, thebags can be readily discarded without disassembly or cleaning. Thedisadvantage of the bags lies in their inherent fragile nature, limitingtheir size; although, significant progress has been made in theconstruction of large disposable bags. Another disadvantage of the bagsis the limited ability to agitate or mix the culture. Linear scale up ofmixing is difficult to sustain with increasing bag size. While the bagvolume increases by the cube, the surface area of the culture head spaceincreases by the square; oxygen transfer becomes limiting as is growthand cell productivity. There are also limitations on monitoring theconditions of the culture with pH, oxygen or other probes, factors whichcan profoundly effect the reproducibility of the culture and limit itsachievable cell concentration and productivity. Considerable progresshas been made by some bag manufacturers to solve the problem ofagitation by incorporating an impeller into the bag, additionally, meansfor sampling, monitoring and making changes to the culture within a bagare being developed; in spite these developments, however, these bagsare used to grow cells in batch or fed batch.

It would be, therefore, desirable to incorporate a device that wouldenhance the productivity of disposable bags or similar systems andalleviate some of their shortcomings. Some desirable features of such adevice may incorporate the following features, including: (i), theability to facilitate mixing of the culture within the disposable bag,(ii), the ability to retain cells and sustain the culture in continuousperfusion mode, (iii), include the capacity to be used externally sothat it may be replaced in mid process with minimum disruption to theprocess, including maintaining process sterility, (iv), remain fully orpartially disposable in nature. One can envision the alternatingtangential flow filtration system described in U.S. Pat. No. 6,544,424as encompassing the above requirements as well as a disposable system,since in its description the device was not limited to constructionmaterial nor to its methods of assembly; however, the system in U.S.Pat. No. 6,544,424 does not describe a system that may be used as acomplete culture system, eg, a system that incorporates or combines theculture vessel and the perfusion device into a single apparatus. Thecurrent invention, as will be described, may be used as such anapparatus that, in addition to providing a means for continuous culture,may be fully disposable, and also offers other benefits and uses.

Changing pressures gradients and flows that are both parallel (axialflow) and perpendicular (transmembrane flow) to the membrane surface areinherent in the alternating tangential flow process. During the“pressure cycle”, the pressure in the pump is greater than the pressurein the retentate reservoir. The retentate flows “forward” from thediaphragm pump, i.e., through the filter element towards the retentatereservoir. Also, some of the liquid is forced across the filter membraneinto the filtrate compartment. Therefore, with an enclosed filtratecompartment, the influx of filtrate can pressurize the filtratecompartment. Conversely, during the “exhaust, cycle” of the alternatingtangential flow filtration process, the pressure in the pump is lessthan that in the retentate reservoir, so that liquid flows in reverse,from reservoir to pump. Additionally, during the exhaust cycle, filtratecompartment fluid pressurized during the previous pressure cycle willalso flow in reverse, from the filtrate compartment to the retentatecompartment. The backflow produces a back flush component that maintainsthe membrane and inhibits clogging. This effect is further enhanced byanother kind of transmembrane flow, one which forms when the resistanceto axial flow inside the hollow fiber, or lumen side, is greater than inthe external, shell, side of the hollow fiber. Therefore, during thepressure cycle pressurized fluid forced into the inlets of the hollowfibers will take the path of least resistance, or in proportion to theresistance, and the fluid will flow not only through the lumens, butalso in part across the membrane, into the filtrate or shell side, aspreviously described. An axial pressure gradient forms on both sides ofthe filter causing fluid flow towards the exit end of the filter.Towards the exit end of the hollow fiber, the lumen pressure decreasestowards its minimum. The pressure drop inside the lumen relative to theshell side results in filtrate reentry into the lumen or retentatestream, on its way to the retentate reservoir. The flow from thefiltrate side back into retentate side provides additional backflushing. It is obvious that such a flow will also be observed duringthe exhaust cycle, but in the reverse direction. The process thusprovides back flushing capacity at both ends of the filter element. As aresult, it is obvious that the described flows offer a great capacityfor exchange of fluids between the retentate and filtrate sides. Suchexchange can be highly beneficial for processing fluids. It is also theobjective of this invention to use this capacity of the alternatingtangential flow filtration system for exchanging fluids across amembrane by modifying the configuration of that system in a manner thatwould result in unique systems and provide great improvement overexisting devices, beyond its previous use as strictly a filtrationdevice.

As will be shown, by specific compartmentalization of the alternatingtangential flow filtration process, one can produce systems that maygreatly improve processing of blood, convert the system into adisposable perfusion bioreactor, facilitate certain biological andchemical reactions or be used for purification or isolation of certainconstituents from biological or other fluids. Other processes and usesare also possible as will become apparent.

BRIEF SUMMARY OF THE INVENTION

The enclosed filtration system invention is a system in which atwo-chambered filter element is enclosed in a chamber, referred to as areactor chamber, such that the reactor chamber and one or both filterelement chambers are accessible outside the system via their ports Thefilter element is preferably a hollow fiber filter cartridge in whichthe internal hollow fiber membranes are semi-permeable and the cartridgeouter wall is semi-permeable or fully permeable. In one embodiment, thispermits intermediate size molecules or complexes to be collected in thefiltrate chamber and even smaller molecules or complexes to be collectedin the reactor chamber. Fluid flow is controlled by an alternating flowpump.

The enclosed bioreactor system comprises 3 types of chambers: filtrationretentate chambers, a filtrate chamber essentially surrounding thefiltration retentate chambers but without blocking their entrance orexit ends, and a processing chamber that essentially surrounds thefiltrate chamber but also does not block the entrances or exits of theretentate chambers. The processing chamber provides an enclosed space inwhich fluid escaping the entrance of the filtration retrentate chambersis captured. The only way fluid from the processing chamber can beaccessed from outside that chamber is through one or more port in theprocessing chamber wall. The filtrate chamber and the filtrationretentate chambers are separated by the semi-permeable membranes of thefiltration retentate chambers, which allow smaller molecules (thosesmall enough to pass through the pores of the membrane) to be moved fromthe filtration retentate chambers and processing chamber to the filtratechamber, from which they can be harvested from the system. Analternating flow pump drives fluid in alternating directions through thefiltration retentate chambers, back and forth from the reservoirchamber.

The manifold invention is a device that is connectable to a fluidsource, such as a bioreactor, fermenter or some other process vessel,and that draws fluid from that source, returns most or all of it to thefluid source; where the direction of fluid flow between source and themanifold is controlled by an alternating flow pump; where the manifoldfurther comprises ports where fluid aliquots can be removed for samplingor for other purposes or added for transport to the fluid source.Optionally, a filter element capable of selecting the fluid's lowermolecular weight substances for testing is part of the manifold;furthermore, the manifold contains further ports through which sensorsmay be added to monitor the fluid flowing within the device; optionally,similar ports with sensors may be added to the manifold to probe thefiltrate within the filter element.

The dual pump system invention comprises two pumps in series, at leastone of them (the first pump) being an alternating flow diaphragm pump.That first pump, will be connectable, via one of its pump chambers, tothe chamber of a bioreactor. That pump will be connected via its otherchamber to a first chamber of the second pump of the system. The secondpump can also be an alternating flow diaphragm pump, or it can be analternating flow mechanical pump. The second pump is connectable to apressure controlling mechanism, generally under electronic power, thatalternatingly creates positive and negative pressures that aretransmitted to the first pump and, via the first pump, to a bioreactorchamber such as a chamber of an enclosed filtration system or anenclosed bioreactor system. Sensors, such as proximity, mechanical,electronic, optical, position or other devices, may be integratedpreferably into the second pump in a manner that indicate the positionof the diaphragm in the second pump. With the use of non-elasticcoupling between the two pumps, the motion of the diaphragm of the firstpump will correspond with the motion of the diaphragm of the secondpump.

The modifier module invention is a module designed for use insidefiltration and bioreactor systems so as to modify some (or lesscommonly, all) of the components in the system. The module, preferablycolumnar in shape, comprises a scaffold and a population of modifieragents, the modifier agent population, that is either homogenous (allagents are the same) or heterogeneous (all agents are not the same).Examples of modifier agents are antibodies or enzymes. The modifieragent population can be bound to the scaffold. In the case where themodifier agent population is not bound to the scaffold, the modulefurther comprises a semipermeable or fully permeable membrane, such thatthe membrane partially or completely surrounding the scaffold andcreates a compartment between the scaffold and membrane, such that whilethe modifier agent population is retained within the compartment,preferably stacked against the scaffold, constituents from the reactoror processing chamber and can pass in one direction or in bothdirections across the membrane to interact with the modifying agent(s).

The product concentration device utilizes a reservoir connected to ahollow-fiber filter element where the reservoir can serve as a containerfor filtrate emanating from another filtering device, such that productin the reservoir can be stored, concentrated and/or further processed asdesired.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a shows a sectional view of an embodiment of the enclosedfiltration system of the invention where the system is connected by aconnector line to a storage vessel, also shown in sectional view, and bya connector line to a compressed air controller.

FIG. 1b shows an enlarged sectional view of the enclosed filtrationsystem of FIG. 1 a.

FIG. 1c is a sectional view of the filter element and filter elementadapter of the enclosed filtration system of FIG. 1b , taken along theline 1 c-1 c in FIG. 1 b.

FIG. 1d is a sectional view of the filter element of the enclosedfiltration system of FIG. 1b , taken along the line 1 d-1 d in FIG. 1 b.

FIG. 1e is a sectional view of the enclosed filtration system of FIG. 1b, taken along the line 1 e-1 e in FIG. 1 b.

FIG. 1f is an enlarged sectional view of a portion of the system shownin FIG. 1 b.

FIG. 1g is a top view of the enclosed filtration system of FIG. 1 b.

FIG. 1h is a partial sectional view of part of the enclosed bioreactorsystem of FIG. 1a . The view enlarges that part of the system ascompared to FIG. 1 a.

FIG. 1i shows a sectional view of an embodiment of the enclosedfiltration system of the invention wherein an adapter is connected to asecond connector line and wherein there is a conduit for fluid recoveryfrom a pump chamber.

FIG. 2 shows a sectional view of an embodiment of the enclosedfiltration system of the invention.

FIG. 3 shows a sectional view of an embodiment of the enclosedfiltration system of the invention.

FIG. 3a shows a sectional view of an example of a enclosed filtrationsystem being used with a dual pump system in which both pumps arediaphragm pumps.

FIG. 3b shows a sectional view of an example of a enclosed filtrationsystem being used with a dual pump system, where the two pumps arediaphragm pumps connected by a connector line controlled by aperistaltic pump.

FIG. 3c shows, in partial cross section and partially in perspectiveview, an example of a pump usable in a dual pump system. The pump is apiston pump driven by a cam mechanism connected to a motor drive shaft.

FIG. 3d shows the pump of FIG. 3c , but with a different position of thecam and coupled piston.

FIG. 3e shows, in partial cross section and partially in perspectiveview, an example of a pump usable in a dual pump system. The pump is apiston pump driven by a reversible screw mechanism.

FIG. 3f shows the pump of FIG. 3e , but with a different position of thescrew and coupled piston mechanism.

FIG. 4a shows a sectional view of an enclosed bioreactor system of theinvention. The reactor chamber of the system is connected to a harvestline

FIG. 4b is an enlarged view in cross section of the system in FIG. 4 a.

FIG. 4c is an enlarged view in cross section of a portion of the systemshown in FIG. 4 a.

FIG. 4d shows a sectional view of a portion of the system of FIG. 4 withan added feature corresponding to a puppet valve.

FIG. 4e is a top view of the system of FIG. 4d

FIG. 4f shows a partial sectional view of an enclosed bioreactor systemof the invention in which the reservoir chamber is horizontallydisposed.

FIG. 4g shows a partial sectional view of an enclosed bioreactor systemof the invention in which the pump is in the upper position and thefilter element is below the pump.

FIG. 5a shows an embodiment of the manifold sampling system invention inpartial cross section.

FIG. 5b shows an embodiment of the manifold sampling system invention inpartial cross-section.

FIG. 6a shows sectional view of a fluid filtration system of theinvention in which the reactor chamber comprises a modifier modulecapable of altering the fluid composition in the reactor chamber.

FIG. 6b shows a sectional view of a modifier module in a storage case.

FIG. 6c Shows a sectional view of modifier module in a storage case

FIG. 6d shows a sectional view of a modifier module.

FIG. 7 shows a sectional view of the enclosed filtration system in whichthe diaphragm pump is separately enclosed.

FIG. 8 shows a sectional view of an embodiment of the system of theinvention for modification or concentration of a cell-depleted filtrate.

DETAILED DESCRIPTION

Terminology

“Fluid” that is processed by the reactor systems of the inventions arenormally aqueous solutions that may or may not comprise suspendedparticulate matter (such as cells, cell fragments, non-soluble molecularcomplexes, particles or soluble molecules). The fluid may or may notcontain molecules that are dissolved in the fluid.

A “selectively permeable barrier” (or selectively permeable wall) is onethat will not allow all particulate matter to pass through it and/orwill not allow all dissolved matter to pass through it.

Normally “selectively permeable barrier” is used herein as a descriptorfor wall of a hollow fiber that is part of a hollow fiber filtercartridge.

A “selective barrier” is another term used herein for a selectivelypermeable barrier. Normally “selective barrier” is used herein as adescriptor for the outer wall of a hollow-fiber filter cartridge.

A fully-permeable barrier (or wall) is one that will permit, at one ormore openings in the barrier, fluid to pass through it without blockingthe passage of any fluid components—either particulate or dissolved.

A fully restrictive barrier does not allow any fluid to pass through it.An opening, such as a harvest port in its open position, must be createdin the barrier to allow fluid to move from one side of the barrier tothe other.

In the enclosed bioreactor system, the outer wall of the hollow fiberfilter cartridge, (i.e., the outer wall of the filtrate chamber) can beselectively permeable, fully permeable, or fully restrictive. Mostcommonly, it is fully restrictive.

Enclosed Filtration System and Process

The enclosed filtration system (and the enclosed bioreactor systemdiscussed below) employs a retentate chamber and a filtrate chamber. Themost convenient way to achieve this is to use a hollow fiber filter.Such a filter is a made as a cartridge that comprises multiple hollowfibers (HF) that run, in parallel, the length of the cartridge and areembedded at each end of the cartridge (preferably with a potting agent);the lumens at the end of the HFs are retained open, thus forming acontinuous passage through each of the lumens from one end of thecartridge to the other, i.e., from a cartridge entrance end, to acartridge exit end. The hollow fibers are enclosed by the outer wall ofthe cartridge (i.e., the cartridge wall) and a potting layer at theirends. As a result, there is a chamber bounded by the cartridge wall andthe outer walls of the HFs. That chamber can be used as the filtratechamber. The intraluminar (internal) spaces of the HFs are consideredcollectively to constitute part of the retentate chamber in each of thepresent systems.

The retentate chamber is extended beyond the internal spaces of the HFsby adapters that fit to each end of the cartridge. Each adapter inconjunction with an end of the cartridge defines a space that is part ofthe retentate chamber. Depending on the direction of fluid flow throughthe fibers, that space serves to either (1) collect fluid as it exitsthe fibers or (2) allow fluid arriving from an external source tointerface with the HF open ends and distribute itself among those HFsfor purposes of continuing its path towards the other end of thecartridge. Each adapter will have two ends, one end fitted to thecartridge and the other end with an opening connectible to a vessel or apump. Normally the vessel is connected to the adapter by a line thatallows fluid flow but, if desired, the vessel can be connected directlyto the adapter or the adapter may form part of the vessel where part orthe entire content of the vessel may be contained within the adapter.Normally the adapter is connected directly to a pump but, if desired,the pump can be connected to the pump via a line that permits fluidflow.

When a connecting line is added to an adapter, the retentate chamber isextended to also include the space inside that connecting line.

When a connecting line is connected at one end to an adapter and at itsother end to a vessel (e.g., one that contains cells suspended in growthmedium), one could consider the interior of the vessel to be a furtherextension of the retentate chamber, but for purposes of description anddiscussion herein the vessel and the retentate chamber are referred toas separate entities.

The walls of the lumens of a hollow fiber filter are permeable,conveniently providing a barrier that is either fully permeable orselectively permeable. The selective permeable hollow fiber walls mayrange in selectivity that ranges the entire gamut of membrane poresizes, commonly classifies asosmotic membranes, ultrafiltrationmicrofiltration to macrofiltration, where, for example, ultrafiltrationrange may encompase Molecular Weight cut-offs, in the range from about10 to about 500 kDa. Pore sizes of 0.2 micron are commonly useful forretaining cells but allowing metabolites and other molecules ormolecular complexes pass through the pores. Pore sizes in the range 10kDA to 500 kDa, are preferred for retaining not only the cells butmolecules and molecular complexes larger than the pore sizes.

The outer walls of filter cartridges are often non-permeable andcommonly have ports from which filtrate can be drained and/or replaced.For purposes of the enclosed filtration system, however, the filtercartridge comprises an outer wall that constitutes a barrier that is maybe non-selective (fully permeable) but is preferably semi-permeable,(not allowing dissolved matter (e.g., molecules and molecular complexes)larger than the pore sizes in the barrier to pass through the barrierand not allowing particulate matter larger than the pore sizes to passthrough the barrier). Pore sizes in the range 10 kDA to 500 kDa, arepreferred for retaining only molecules and molecular complexes largerthan the pore sizes. However, the pore sizes can be made small enough orlarge enough. so that, respectively, the barrier is highly restrictive,only allowing small salts and their components to pass through orallowing molecules or particles larger than 500 kDa to pass through themembrane. Such membrane selectivity are not only restricted to pore sizebut to other membrane properties, including: charge, hydrophobisitymembrane configuration, membrane surface and pore polarity, etc.

The enclosed filtration system, in its general aspect, is a system thatcomprises:

-   -   1) a retentate chamber, said retentate chamber comprising an        entrance at its entrance end and an exit at its exit end, said        retentate chamber comprising a retentate chamber wall, at least        a portion of said wall being semi-permeable;    -   2) a filtrate chamber (e.g. the portion of the inside a hollow        fiber filter cartridge that is external to the hollow fibers),        said filtrate chamber at least partially enclosing said        retentate chamber, said filtrate chamber comprising a filter        chamber inner wall and a filter chamber outer wall, wherein at        least a portion of the filter chamber inner wall corresponds to        the semi-permeable portion of the retentate chamber wall; said        filtrate chamber outer wall comprising a filtrate chamber outer        barrier (possibly fully permeable, but preferably        semi-permeable);    -   3) an alternating flow pump, said pump said pump connected to        the perimeter of the retentate chamber exit so as to permit        fluid from the pump to enter the retentate chamber and fluid        from the retentate chamber to flow into the pump; said pump        comprising an outer wall, a diaphragm, and two chambers        separated by the diaphragm;    -   4) a reactor chamber, said reactor disposed so that it at least        partially encloses both the filtrate chamber and the retentate        chamber in a sealed manner but does not block fluid flow in and        out of the retentate chamber entrance, said reactor chamber        comprising a reactor chamber inner wall and a reactor chamber        outer wall, said reactor chamber inner wall comprising the        filtrate chamber outer barrier, said reactor chamber outer wall        being sealed to the outside of either the retentate chamber or        to the outside of the filtrate chamber, said reactor chamber        outer wall optionally sealed to the alternating pump outer wall;    -   5) a harvest port, said harvest port attached to the reactor        chamber outer wall so as to allow fluid to leave or enter the        reactor chamber.

As indicated above, one can access the filtrate reservoir through one ormore ports to remove filtrate or to make additions to the filtrate inthe filtrate chamber.

The terms “sealed”, “sealingly” and the like refer to the fact that thejuncture or junction of two chambers or other systems components doesnot permit fluid to leak through the juncture or junction.

The foregoing enclosed filteration system and the variant below wherethere is a filtrate reservoir are exemplified in the Figures and thedetailed discussion of the Figures below.

The enclosed filtration system, in an aspect referred to as the“filtrate reservoir system”, may further comprise a filtrate reservoirconnected to the filtrate chamber, such that fluid flow directly betweenthat reservoir and the filtrate chamber is permitted. Here, the reactorchamber outer wall is sealed to the outside of the filtrate chamber, butdoes not enclose the portion of the filtrate chamber that opens into thereservoir. The reservoir encloses the portion of the filtrate chamberthat opens into the reservoir. The reservoir is separated from thereactor chamber so that there is no fluid exchange directly between thereservoir and the reactor chamber. In addition to the harvest/additionport(s) connected to the reactor chamber (the first set of port(s)), thesystem comprises a second set of port(s) which connect to the filtratereservoir and which extends outside the filtrate reservoir.

The size of the pores in the filtrate chamber outer barrier may bevaried depending on the intended use of the system.

In both the enclosed filtration system and the filtrate reservoirsystem, there may be a second harvest port, that port attached to thefiltrate or reservoir chamber so as to allow fluid to leave or enter thefiltrate or reservoir chamber.

The diaphragm pump contains two pump chambers and a diaphragm inbetween. One pump chamber is connected to the retentate chamber so thatdirection of fluid flow through the retentate chamber may be confine andcontrolled by the pump. The other pump chamber, the drive chamber, isconnected to a source of alternatingly positive and negative pressure.

For the enclosed filtration system and the filtrate reservoir system, a(conduit) connector line will normally be connected to the diaphragmpump's externally connected chamber, or drive chamber, as part of theconnection between that chamber and the pressure controller.

For the enclosed filtration system and the filtrate reservoir system, aconnector line will normally be connected to the retenate chamber'sport. For the enclosed filtration system and the filtrate reservoirsystem, the line will serve as part of the connection to the externalbioreactor, vessel or other containers of retentate.

For the enclosed filtration system and the filtrate reservoir system,additional connector lines and/or harvest lines are used as needed.Many, but not all, such possibilities are illustrated by the examplesherein.

Additionally, the enclosed filtration system and the filtrate reservoirsystem may further comprise additional ports to the retentate, filtrateand/or reactor compartments: for the insertion of probes, instruments ordevices, and/or for making additions and subtractions of substances.

Like the alternating tangential flow filtration system, the enclosedfiltration system and the filtrate reservoir system provide uniform flowthrough the entire filter. Also like the alternating tangential flowfiltration system, the enclosed reactor systems of the invention may beused with a variety of filter types.

A device that combines the filtrate chamber and the retentate chamber isreferred to herein as a “filter element”. One filter element that may beused is a hollow fiber (HF) filter, whose use is extensively describedherein. They are available in many sizes, configurations, materials,pore sizes, porosity and housings. However, the systems of the inventiondo not require the use of hollow fiber filters. It is possible toutilize other separation devices. One such device may be a “plate andframe” filter. Another device is a screen module, consisting of a screenmesh as the separation membrane.

It can be seen that the reactor chamber may be used for a variety offunctions. For example, it may be used as a permanent or temporarystorage reservoir. This makes the content available for variousmodifications or processing prior to its return to the main process orprior to its harvest.

The enclosed filtration reactor systems of the present inventions (andthe enclosed bioreactor systems described below) can be used inadditional applications including but not limited to, kidney dialysis,blood processing, water purification concentration, fluid exchange, orvarious other filtration applications. To illustrate, in the case ofkidney dialysis such a situation, the patient's circulatory system canbe connected to the retentate chamber. The reactor chamber and/or thefiltrate chamber is the source of particular fluid components(electrolytes, biologically active components, adsorptive agents andothers) at the concentrations or volumes desired to facilitate thedialysis process. Due to the inherent “lateral” flow during thealternating tangential flow process, fluxes between chambers facilitaterapid equilibration between the compartments. The selectively permeablebarrier between the retentate and filtrate chambers may provide onerestrictive barrier. Optionally, selective barrier between the filtratechamber and reactor chamber offer a second restrictive barrier;therefore, the combination of selectivity and rapid equilibrationbetween compartments process offers a more efficient process for removalof undesired or toxic byproducts from circulation i.e., those smallenough to pass from the retentate into the filtrate chamber and/orreactor chamber.

It is desirable that enclosed reactor systems of the invention besterilizable and that, subsequent to sterilization, can be storedsufficiently sealed to prevent subsequent contamination of its interiorby external microorganisms or other contaminants that otherwise wouldenter into the interior of the system subsequent to its sterilization.It is also desirable that these systems are in a configuration and madewith construction materials that render the system disposable.

Processes of the Invention Using the Enclosed Filtration Systems

The enclosed filtration system process, in a general aspect, comprisesthe steps of:

-   -   1) discharging fluid from a retentate chamber via a fluid        connector into a vessel (such as a storage vessel) such that        during said discharging a portion of said fluid is directed via        a semipermeable barrier into a filtrate chamber and is then        directed via a selective barrier into a reactor chamber, wherein        said discharging is due to the force exerted by a diaphragm pump        connected to the retentate chamber at a position distal to the        fluid connector; and    -   2) Reversing the direction of the force exerted by the diaphragm        pump so that at least some fluid from the vessel flows back into        the retentate chamber and at least some fluid from the retentate        chamber flows into the filtrate chamber (and preferably some        fluid from the filtrate chamber flows into the reactor chamber);        and    -   3) Repeating steps (1) and (2) at least once, wherein fluid        discharged from the retentate chamber is selected from the group        consisting of a suspension and a solution, and wherein the        retentate chamber, filtrate chamber, reactor chamber, and        diaphragm pump are part of the same enclosed filtration system        (preferably wherein the enclosed filtration system is described        in the general aspect or second aspect herein above).

In the foregoing and following process, the fact that fluid crosses aportion of a barrier in one direction does not preclude, and indeed isoften associated with, fluid flow in the opposite direction at anotherportion of the barrier.

Normally, material from the retentate, reactor chamber and/or thefiltrate chamber will be harvested at least once.

In a variation of the enclosed filtration system process, applicable toa system that comprises a filtrate reservoir, the process comprises thesteps of:

-   -   1) discharging fluid from a retentate chamber via a fluid        connector into a vessel such that, during said discharging, a        portion of said fluid is directed via a selectively permeable        barrier from the retentate chamber into a filtrate chamber, such        that a portion of said fluid directed into the filtrate chamber        is directed via an opening in the filtrate chamber wall to a        filtrate reservoir and such that a portion of said fluid        directed into the filtrate chamber is directed via a selective        barrier into a reactor chamber, wherein said discharging is due        to the force exerted by a diaphragm pump connected to the        retentate chamber at a position distal to the fluid connector;        and    -   2) Reversing the direction of the force exerted by the diaphragm        pump so that at least some fluid from the vessel flows back into        the retentate chamber, at least some fluid from the retentate        chamber flows into the filtrate chamber, at least some fluid        from the filtrate chamber flows into the filtrate reservoir and        at least some fluid from the filtrate chamber flows into the        reactor chamber; and    -   3) Repeating steps (1) and (2) at least once, wherein fluid        discharged from the retentate chamber is selected from the group        consisting of a suspension and solution, and wherein the        retentate chamber, filtrate chamber, reactor chambers and        diaphragm pump are part of the same filtrate reservoir system,        (preferably wherein the filtrate reservoir system is described        herein above).

Normally, material from the retentate, filtrate reservoir and thereactor chamber will be harvested at least once.

FIGS. 1a-1h illustrate various views of the enclosed filtration systemand devices connected to it. (A reference number (e.g., 45 for theretentate chamber) used in one of those views is applicable to the samecomponent in all other of those views.)

In FIG. 1a , an enclosed fluid filtration system 1 is connected via afluid connector 3 to a process vessel 2, containing the fluid materialor retentate 9 to be processed. (In FIGS. 1a, 1b and 1h , the number 9indicates the location of retentate, not the retentate itself (exceptfor the retentate fluid shown in vessel 2), in the event that system isin use and contains retentate). The fluid filtration system 1 containsat least three chambers: a retentate chamber 45 (shown in greater detailin FIGS. 1b-1h ) confining the unfiltered material, a filtrate chamber10 (shown in greater detail in FIGS. 1b-1h ) within the filter element 5(for example, a hollow fiber filter) and a reactor chamber 11 separatedfrom the filter element by a selectively permeable barrier 19 (forexample, a hollow fiber cartridge's outer wall or another membrane).

The fluid filtration system 1 is enclosed by housing 15, whose shape,size or orientation may be varied as needed to enclose the system. Thehousing 15 may be constructed from a variety of materials, includingsolid polymers, such as polycarbonate or polysulfone, flexible orelastic materials or any other material or composite of materials. Theprocess vessel 2 may be any suitable container for a fluid to beprocessed. For example, it may be a bioreactor, a circulatory system orany other vessel, nonexclusively including tanks, bags, flasks and thelike which can contain liquids. The process vessel 2 may be composed ofany suitable material or combination of materials, including, syntheticpolymers, inert metals, such as stainless steel, glass, etc; nor shallthey exclude rigid, flexible or elastic materials or a combinationthereof; nor should such materials be limited in shape, size orconfiguration, as long as they result in a process vessel. The processvessel 2 is not limited as to accessibility: It may be modified to allowadditions to or subtractions from the content of the vessel. Lines ortubes 36 and 39, for example, can be used to effect additions to orsubtractions from the content of process vessel 2, for example using apump 14 to control such addition or subtractions. Such process vesselsare commercially available in all sizes and configurations, and are wellknown to those in the field. The fluid connector 3 serves to direct afluid from the process vessel 2 via fluid exchange port 35 to theentrance end 42 of filter element 5 which also corresponds to theentrance end of the retentate chamber 45. Entrance 42 while serving asan entrance to chamber 45, may also serve as a reservoir for retantate;its shape and positioning may be varied according to need; its volumemay be approximately equal to the diaphragm pump displacement volume,facilitating between reservoir 42 and pump, and further facilitatinggreater level of retantate concentration and recovery of finalconcentrate. (FIG. 1b ; The construction of the hollow fiber filter isbest understood by also taking into account FIGS. 1b through 1h .) Theport 35 is held in place by entrance end adapter 40 which may also serveas the end cover for entrance end reservoir 42, which in turn serves asthe cover and entrance end to filter element 5; in combination, 35, 40and 42 serve as a conduit adapter through top plate 16 of housing 15 fordirecting fluid to the filter element entrance end 42. As a reservoir,reservoir 42 it may be placed above, through or below top plate 15 andits positioning and configuration would not interfere with ports fromthe filtrate chamber(s) or reactor chamber(s). The fluid flow is furtherdirected through the filter channels 17, which would correspond to theinteriors of the lumen(s) of a hollow fiber filter should filter element5 correspond to a hollow fiber filter. The filter channels collectivelycorrespond to the retentate chamber 45 of the fluid filtration system 1.In one direction, the fluid flow proceeds to, and exits from, the exitend 43 of the filter element 5 Adapter 41 at the exit end 43 of both thefilter element 5 and the retentate chamber 45 directs the fluid from thefilter element exit end 43 to the liquid receiving chamber 7 of adiaphragm pump 4. Adapter 41 is not limited by its shown configurationor shape; it may be connected to pump 4 directly or through a pumpadapter 29; or it may connect the filter exit end 43 to pump 4 through aconduit (not shown here).

The flow through the filter element 5 between process vessel 2 anddiaphragm pump 4 (an alternating flow pump) is generated by pump 4 aspreviously described in U.S. Pat. No. 6,544,424. Pump 4 preferablycomprises a pump housing 4 separated into a first interior chamber 8,also referred to as “the first chamber” or a “drive chamber”, and asecond interior chamber 7, also referred to as the “second chamber” or_“the liquid receiving chamber”, by an internal diaphragm 6. The pumphousing 4 in FIGS. 1a and 1b is made of two housing components, thefirst pump housing component 25 and the second pump housing component24. The components comprise flanges 26 and 27, respectively. Pressure inthe first interior chamber 8 drives the diaphragm within diaphragm pump4 without causing contamination of the fluid content in the secondinterior chamber 7. Shown in FIG. 1a is an air driven pump. Compressedair is directed by controller 54 to chamber 8 through line (tube) 21,preferably through a sterilizing filter 22 and an air inlet port 23.Increasing the air pressure in chamber 8 relative to process vessel 2drives a flexible diaphragm 6 into chamber 7, driving liquid in thatchamber through the filter element 5 to vessel 2. The reverse flow fromprocess vessel 2 to pump 4 is generated by reducing the pressure inchamber 8 relative to the vessel 2. The cycles are repeated. Alternatingflow generated by such a pump has been described in U.S. Pat. No.6,544,424.

Facilitating the construction of an enclosed filtration system, it maybe preferable to fix the flexible diaphragm outer flange 47 (FIG. 1f )to the perimeter of the pump housing 26 and 27 (FIGS. 1b and 1f ) with aleak proof connection; typically by using a clamping mechanism tosqueeze the diaphragm between the flanges 26 and 27 of the two pumphalves, which pump halves comprise chambers 7 and 8. While suchconnection may be accomplished in a number of ways, an example is shownin FIGS. 1b and 1f ; as illustrated, peripheral flanges 26 and 27 on therespective diaphragm pump halves contain an “O” ring groove 28, which isdesigned to accept the counterpart “O” ring segment 44 on the flangeportion of diaphragm 6. A preferred embodiment of the peripheral flanges26 and 27 and “O” ring groove 28 is one that permits bonding theperipheral flanges 26 and 27 together and simultaneously secure thediaphragm “O” ring flange 47 and “O” ring segment 44 between flanges 26and 27; this may be accomplished, by controlling the spacing betweenadjacent faces of the flanges 26 and 27, as follows: From the outerperimeter of the diaphragm 6 inwards, towards the center axis of thediaphragm pump 4, the diaphragm pump flanges 26 and 27 are spaced fromeach other by a distance somewhat less than the corresponding thicknessof diaphragm 6 flange segments 47 (i.e. the flange segments of thediaphragm), including its “O” ring segment 44 (FIGS. 1b and 1f ). Thatspacing between the flanges minus the thickness of the diaphragm istermed “compression distance”. From the outer perimeter of the diaphragm6, outward, to the outer perimeter of flanges 26 and 27 the spacingbetween the facing surfaces of flanges 26 and 27 is equal to thecompression distance; therefore, when the two facing flanges 26 and 27are forced together, they compress, by the compression distance, thediaphragm flange segments 47 and the “O” ring segment 44, at the sametime forming a contact surface 48 between the two flange segments 26 and27. Once in contact, peripheral flanges 26 and 27 can be bonded to eachother along their contact surface 48; in the process, diaphragm 6 issealed securely between the pump segments. The method allows for settingthe magnitude of the compression on diaphragm 6 flange 47 and “O” ringsegment 44 by controlling the compression distance between correspondingand adjacent pump flange segments. Further, to assure that the flangesof the two diaphragm pump halves remain bonded securely along bondingsurface 48, the bond can be further reinforced along the surface 49 offlanges 26 and 27. This is exemplified in FIG. 1f by securely bondingflange 26 and 27 ends against the inner wall of the housing 15, forminga bond along surface 49, preferably in a leak proof manner. Theprocedure results not only in securing the diaphragm within the pump andin securing the pump to a confining scaffold, but also in other uniquebenefits. One such benefit involves using a cylindrical housing 15 thatextends the length of the fluid filtration system 1 to form an enclosurefor the entire system, including all its vital components. Thecylindrical housing 15 may serve as a stand for the entire system, tomaintain the system either in the upright position (as shown) or upsidedown position The bonding or securing of the peripheral flanges 26 and27 to the housing 15 adds structural support to the entire system toprotects its content. Another benefit is the formation of a “base” 25,FIG. 1b , to the reactor chamber 11. In addition, by providing theenclosure 15 with a top 16, its possible to fully enclose the filterelement 5 and the reactor chamber 11. Enclosure 15 and top 16 may beconfigured to a desired form and designed to accept various elements,accessories or insertions to make additions and or subtractions tochamber 11, to monitor or affect conditions within; all of which, aswill be shown, increase the system's versatility. FIG. 1a shows anexample of an enclosure 11 formed by wall 15, base 25 and top 16; shownalso is a system containing a filter element 5 connected at one end tothe pump 4 and at the other end to adapter 40, which forms a passagewayto conduit 3 and through enclosure top 16. With top 16 and pump flanges26 and 27 bonded or sealed against enclosure 15, the filter element 5 ispositioned within the reactor chamber 11, where the filtrate chamber 10(and the filter element 5) share a common selective barrier 19 withreactor chamber 11 as part of their respective walls. (Filtrate chamber10 is defined by the surfaces of the lumens 17, by the cartridge outerwall 19, and by the areas filled with the potting material 44). Bycontrolling the properties of selective barrier 19 a highly usefulselective barrier can be formed between reactor chamber 11 and filtratecompartment 10 (and thereby with filter element 5). As will be shown,using the well known function of the filter element 5 and its capacityfor selective separation of constituents between the filtrate chamber 10and the retentate chamber 45, and further providing a selective barrier19 between filter element 5 and reactor chamber 11 results in a uniquedevice with multiple useful functions.

The harvest from reactor chamber 11 is collected via line 13 which isconnected to harvest port 12, which allows fluid exchange between thechamber and that line so as to allow fluid to enter or leave the reactorchamber. (See also FIG. 1b for the relationship of harvest port 12 (orits equivalent) through top 16 (or its equivalent).)

The housing 15, diaphragm pump 4, diaphragm 6, valves, filters and otherconstituents of the system may be constructed from various materials,preferably from materials that withstand the pressures generated duringoperation of the fluid filtration system 1, preferably, from materialsthat may be sterilized either chemically, with steam or radiation; forexample, such materials may including stainless steel. However, one ofthe primary disadvantages of stainless steel is the inability to viewthe content inside housing 15 or inside the diaphragm pump 4. Some otherdisadvantages of stainless steel is weight, cost, and difficulty to forminto specific shapes. It is therefore preferable, particularly when adisposable system is required, to use materials such as, polycarbonate,polysulfone or others, that are selected for their structural strengthand transparency; such materials, that can be readily molded intodesired shapes, that are light and relatively inexpensive, that can alsobe sterilized chemically, with radiation or with steam. An additionaldesired features of a construction material is its suitability tomanufacturing techniques, its amenability to its packaging, storage,transportability, and to providing protection against damage orcontamination.

FIGS. 1a-1h illustrate a hollow fiber filter 5 useful for the presentinventions. (The structural features visible in FIG. 1h are more easilyseen than the same features as shown in FIG. 1b .) The filter 5 is amade as a cartridge that comprises multiple hollow fibers (HF) that run,in parallel, the length of the cartridge, from a cartridge entrance endto a cartridge exit end. A segment of the hollow fibers are externallypotted at both ends of the cartridge, by methods common to manufacturersof hollow fiber cartridges; the hollow fibers are enclosed by the wallof the cartridge and the potting material 44 at the ends of thecartridge. Examples of potting compounds are epoxies and polyurethanes.As a result, there is a chamber bounded by the cartridge wall 19 pottedends 44 and the walls 17 of the HF. That chamber can be used as thefiltrate chamber for the present inventions. One could say that thereare multiple retentate chambers, each corresponding to a single HFintraluminar space. However, for purposes of description, theintraluminar spaces are considered collectively to constitute aretentate chamber in each of the present systems.

The walls 17 of the lumens (hollow fibers) of the illustrated hollowfiber filter are selectively-permeable, conveniently providing theselectively permeable wall referred to in the descriptions of thesystems. The outer wall 19 of the filter cartridge (the cartridge wall)comprises a selective barrier referred to in the descriptions of thesystems, that barrier also being selectively permeable.

FIG. 2 also illustrates the enclosed filtration system 1 of FIGS. 1athrough 1h but shows it augmented with coverings 50 and 51. In FIG. 2,the enclosed filtration system 1 is connected to both a connector line 3(i.e., a fluid connector line) and, via harvest port(s) 12 to harvestline(s) 13. Furthermore the system is enclosed by coverings 50 and 51 ateach end, so that its otherwise exposed ends are fully enclosed. Thefluid connector line and harvest line(s) are shown as folded because inthis particular example they need to be folded so that they can beenclosed by the coverings. Fluid connector line 3 and harvest line(s) 13are each protected with a shield 52 at their end.

Reference numbers 4, 5 and 15 in FIG. 2 apply to the same componentsthey refer to in FIGS. 1a through 1 h.

FIG. 3 illustrates a variation of the enclosed filtration system 1 shownin FIGS. 1a-1h . The system 501 shown in FIG. 3 has filtrate reservoir564 not present in the system FIGS. 1a-1h . In FIG. 3, filtratereservoir 564 and the reactor chamber 511 are separated from each otherby a separating barrier 560 that does not permit direct fluid exchangebetween the reservoir 564 and the chamber 511. Both the filtratereservoir 564 and the reactor chamber 511 can undergo fluid exchangeseparately with the filtrate chamber 510 of the filter element 505.Fluid exchange between the filtrate chamber 510 and the reactor chamber511 is effected through a portion of the filter element 505, which hasan outer wall similar in structure to the outer wall of the filterelement 515 in FIGS. 1a-1h ; i.e., it is a selective barrier.

In contrast, fluid exchange between the filtrate chamber 510 and thefiltrate reservoir 564 is effected through an opening 561 which providesa direct conduit from the filtrate chamber 510 to reservoir 564, wherefiltrate can be harvested via harvest port 562 (“the second harvestport”) and line 563. The harvest from reactor chamber 511 is collectedvia line 513, which can access reactor chamber through tube 512 thatextends into that chamber; functioning as a port (“the first harvestport”).

In the system 501 in FIG. 3, the housing 515 is similar to the housing15 in FIGS. 1a and 1 b.

In FIG. 3, critical connections, including the fluid connector segment503, harvest tubes 512, 562, pump 504, filter element 505, and othersystem accessories, in their various forms, are shown enclosed and fullyprotected by coverings 550 and 551. Such coverings may also encloseother lines or devices connected to the system or to its variouscompartments. These may include tubings, sensors, electric lines,filters etc.; such devices may be further enclosed or protected bysecondary shields; for example, fluid connector segment 503 and harvesttubes 563, may be further protected with shield 552 at their end;therefore, when covering 550 is removed and fluid connector segment 503and harvest lines 563 are exposed, their internal volume remainsprotected against contamination while remaining available for theirconnection to their counterparts in a sterile manner.

It can be seen however, that many other components and features of theenclosed filtration system 1 as described in FIGS. 1a-1h are present insystem 501 in FIG. 3. For example, all components of the filter element5 are essentially paralleled by the filter element 505 in FIG. 3. Alsothe diaphragm pumps 4 and 504, and the interconnection between thefilter element and the pump, are not only present in FIGS. 1a and 1b butas well as in FIG. 3. Such common components further include but are notlimited to the retentate chamber 45 and 545 and the selectivelypermeable barrier 19 and 519). Furthermore fluid exchange port 35 ofFIGS. 1a-1h is present is present in FIG. 3.

The enclosed fluid filtration system and filtrate reservoir system shownin FIGS. 2 and 3 may each be encapsulated in a protective bag foradditional protection against contamination or mishandling; the entiresystem may be sterilized at once. Such safety features are essential inforeseen applications such as dialysis as well as other medical and cellculture related applications. In FIGS. 1a-1h and, as applicable, FIGS. 2and 3, the primary function of the fluid connector 3 is to provide areliable, sterile, low sheer fluid conduit. Preferably, it should allowthe flow to be bidirectional between process vessel and fluid filtrationsystem 1; furthermore, when using the system as a disposable unit, itbecomes essential that a reliable and simple connection is formedbetween such a disposable unit and the process vessel. A preferredfeature of the fluid connector 503 is the ability to form, break, orreform the connection between the fluid filtration system system 1 andthe process vessel 2 in a sterile and reliable manner. There are anumber of well known techniques for joining or breaking a fluidconnector in a sterile manner, including the use of tube welders,SIPable valve assemblies, joining or uncoupling sterilized couplingswithin a biological safety hood, use of sterile connectors such as theclean-pack (by Pall corp.) or the DAC™ (by GE), or for that matter anyother device which permits a sterile connection or disconnection betweentube segments; such connections, however, do not exclude using fluidconnectors that may include more than one fluid connector, where one ormore connector serve to deliver fluid to the enclosed filtration systemand one or more connectors serve to deliver fluid from the enclosedfiltration system; such connector(s) may include assorted types ofvalves, couplings, or devices which affect the flow through the conduit,including check valves, sensors, restrictors, etc. The fluid connectormay also be partitioned to access more than one port on the processvessel or multiple vessels or multiple enclosed reactor systems, as theneed arises. The fluid connector may also incorporate probes, such asflow meters, for monitoring flow rates through the connector, probes,the likes of pH, dissolved oxygen, etc., for monitoring the vitality ofthe culture flowing through the fluid connector. The rapid flow betweenvessel and enclosed reactor system reflects the conditions in theenclosed reactor system and process vessel. Any combination of the aboveor similar modifications to the fluid connector may be used by thosewith knowledge in the field.

For the enclosed filtration system and the filtrate reservoir system, aconnector line will normally be connected, via a retentate chamberentrance end adapter to the entrance end of the retenate chamber. In anembodiment of the enclosed filtration system, referred to as themodified adapter embodiment, the system further comprises:

-   -   1) A reservoir adapter as its retentate chamber entrance end        adapter, such that said reservoir adapter, in addition to being        connected to a first connector line also comprises a second        connector line;    -   2) a drainage tube connected at one of its two ends to the        entrance chamber of the alternating flow pump, said drainage        tube extending to a point exterior to the enclosed filtration        system so that retentate in the entrance chamber of the pump can        be collected outside the system.

An example of the modified adapter embodiment of the enclosed filtrationsystem is illustrated in FIG. 1i . It can be seen that the FIG. 1i canbe considered a modified version of the enclosed filtration systemillustrated in FIGS. 1a -1 h.

Here the system comprises a reservoir adapter 42. In addition toconnector line 71 that is connected to the reservoir adapter, there is asecond connector line 73 connected to the reservoir adapter. Connectorline 71 can be connected to an outside source of fluid such as a vesselbut is particular suited for being connected via a catheter to aperson's blood s stream, in which case the connector line will also beconnected to the blood stream. The blood will enter the filtrationsystem via line 71 and exit via line 73. In that mode, the system issuited for blood and kidney dialysis.

In the dialysis mode, an advantage of the system is that the benefits ofan alternating tangential flow filtration system are utilized but theblood stream in the patient is always in the same direction.

When line 71 is connected to a vessel outside the system (such as vessel2 in FIG. 1a ), the modified adapter embodiment is also well suited torecovering small volumes from the vessel if such small volumes occurbecause of continued titration.

In the modified adapter embodiment, one may add a pressure sensor to thereservoir adapter.

It can be seen from FIG. 1i that the embodiment also comprises adrainage tube 60, 62, which can be considered to have two sections, 60and 62, respectively. The drainage tube is available for recovery ofretentate, especially the final retentate volume, and generally forsampling the retentate. It is also available for diluting the retentate.

The volume of the reservoir adapter is preferably equal or slightlylarger that the displacement volume of the diaphragm pump 4, but it canbe any size.

Pumps 72 and 74 allow control of fluid addition to the reservoir adapterand the rate of removal of modified (concentrated) fluid from thereservoir adapter. The two pumps may be used as valves in a manner thatallows pressurization of reservoir adapter 42. The system can thus bepressurized (requiring no vacuum for operation), which is useful formany filtration applications.

A diaphragm pump (“the reservoir pump”) may be used in place of thereservoir adapter, in which case the system will comprise two suchpumps. The reservoir pump would have two connector lines connected toone of its chambers. The other chamber would be connected to the filterelement. The diaphragms of the two pumps would have to move insynchrony.

The features of the enclosed filtration systems facilitate their rangeof applications, storage and transportability. As shown in FIGS. 2 and3, for example, the entire system may be packaged in a self-containedcontainer. The pump(s), filter, fluid connector, harvest line, modifiermodule, modifier suspension solution can all be conveniently providedpreassembled, enclosed and sterile.

An important attribute of the invention is the closed nature of theenclosed filtration system (as it is for the enclosed bioreactor systemsdescribed below). The enclosed nature of the system also allowsfiltration applications with hazardous materials (i.e., corrosive,flammable, bio hazardous, etc.), provided the appropriate filters andall other components of made of materials that are compatible with theprocess. This may include the use of filters made from metals, ceramicsor other material. Similarly, the diaphragm and other components of thesystem may be made from any number of materials that will allowcompatibility with the requirement of the process. As was described, allcomponents can be connected in such a manner as to totally confine theprocess.

Enclosed Bioreactor System and Process

The enclosed bioreactor system, in a preferred aspect, comprises thefollowing:

-   -   1) a hollow fiber filter element (preferably a cylindrical        hollow fiber filter cartridge) said filter element comprising an        entrance end and an exit end, said filter element further        comprising a plurality (more than one) of filtration retentate        chambers, each filtration retentate chamber being an open-ended        hollow fiber, said fibers disposed in parallel to the center        axis of the filter element (the center axis extends through the        center of the cylinder from one filter element end to the        other), wherein the fibers each have an entrance at the entrance        end of the filter element and an exit at the exit end of the        filter element, and wherein each fiber comprises a        semi-permeable outer wall, said filter element further        comprising a filtrate chamber that encloses said fibers but does        not block their open ends, such that the semi-permeable outer        walls of the fibers are also part of a filtrate chamber wall;    -   2) a filtrate harvest tube (preferably rigid), said tube        penetrating the filter element and its filtrate chamber via the        filter element entrance end, said tube, preferably, disposed        along the center axis of the filter element;    -   a, an alternating flow diaphragm pump, said pump connected to        the exit end of the filter element so as to permit fluid from        the pump to enter the filtration retentate chambers, said pump        comprising a pump housing, two chambers, and a diaphragm        separating the chambers,    -   4) a processing chamber, said chamber enclosing the cartridge,        said chamber comprising a base, an outer wall (preferably        cylindrical), a base, and a top plate, wherein the base is        attached to both the outer wall and the pump housing in a sealed        manner and wherein said top plate is attached to the outer wall        in a sealed manner, and wherein the top plate is penetrated by        the harvest tube so that fluid can flow from the titrate chamber        to outside the reservoir chamber;    -   5) A processing chamber harvest line or lines and a processing        chamber addition line or lines, said harvest and addition lines        penetrating either the top plate (preferred), outer wall, or        base of the processing chamber so that fluid can be harvested or        removed from the processing chamber or added to it;    -   6) A port in either the top plate (preferred), outer wall, or        base of the processing chamber through which oxygen can be fed        into the processing chamber, said port comprising a sterilizing        filter to prevent contamination of the processing chamber by        microorganisms; and    -   7) Port(s) in either the top plate (preferred), outer wall, or        base of the processing chamber through which sensing devices can        be inserted into the processing chamber.

Preferably the foregoing preferred aspect of the enclosed bioreactorsystem comprises a tubular fluid connector inside the processing chamberand, preferably but not exclusively, disposed around the filter elementsuch that said connector is not in direct contact with said filterelement, said connector comprising a sealed exit end or sealed end andan entrance end, said sealed end disposed between the top plate of theprocessing chamber and the entrance end of the filter element, saidsealed end penetrated by the harvest tube, said sealed end fordeflecting fluid flowing from the filter element entrance so that suchfluid flows through a separation space separating the fluid connectorand the filter element, said fluid connector comprising an open endthrough which the deflected fluid can escape into the processingchamber;

It is preferred that the enclosed bioreactor system comprise a pluralityof sparger holes in its base, which holes function as part of the portsthrough which the oxygen is fed into the processing chamber

It is also preferred that the enclosed bioreactor system compriseadditional ports, in either the outer wall, top plate, or base of thereservoir chamber so that fluid (or fluid containing suspended cells),can be added to the processing chamber

It is also preferred that enclosed bioreactor system comprise anagitation device; exemplified by an open-ended draft tube (open at bothends; preferably cylindrical) inside the reservoir chamber andsurrounding all or a part of the fluid connector tube, said draft tubenot directly touching the fluid connector tube, said draft tube held inposition by a support frame also connected to the outer wall of thereservoir chamber.

The exit of the pump is preferably protected by a filter that preventsmicroorganisms from entering the pump.

In a particular embodiment, the sealed end of the fluid connector tubeis modified so that it comprises a puppet valve the permits part of thefluid to move in one direction directly from the processing chamber intothe space between the valve and the entrance end of the filter element.However, the puppet valve does not permit fluid to flow in the oppositedirection, deflecting fluid flowing from the filter element entrance sothat such fluid flows through a space separating the tube and the filterelement, directing the fluid exiting from the entrance end of the filterelement to the processing chamber.

An enclosed bioreactor system comprising, in a general aspect, thefollowing:

-   -   1) a filter element, said filter element comprising an entrance        at an entrance end and an exit at an exit end, said filter        element further comprising a plurality of filtration retentate        chambers (such as a plurality of hollow fibers) wherein the        filtration retentate chambers each comprise an entrance at the        entrance end of the filter element and an exit at the exit end        of the filter element, each of said filtration retentate        chambers further comprising an outer wall, each said outer wall        comprising a semi-permeable portion, said filter element further        comprising a filtrate chamber that encloses the semi-permeable        portions of the filtration retentate chamber outer walls but        does not block the exits or entrances of the filtration        retentate chambers, such that the semi-permeable portion of the        outer walls of the filtration retentate chambers is also part of        a filtrate chamber wall;    -   2) a filtrate harvest tube, said tube penetrating the filter        element and filtrate chamber so as to permit fluid to leave the        filtrate chamber;    -   3) an alternating flow pump, said pump connected to the exit end        of the filter element so as to permit fluid from the pump to        enter the filtration retentate chamber, said pump comprising a        pump housing, two chambers, and a diaphragm separating the        chambers,    -   4) a processing chamber, said chamber enclosing the filter        element, said chamber comprising an outer wall, said outer wall        attached to the pump housing in a sealed manner, wherein said        outer wall is penetrated by the harvest tube so that fluid can        flow from the titrate chamber to outside the reservoir chamber;    -   5) A processing chamber harvest and addition line(s), said        harvest and addition line(s) penetrating the wall of the        reservoir chamber so that fluid can be, respectively, harvested        or removed from the processing chamber or added to the        processing chamber;    -   6) A port in either the wall of the reservoir chamber through        which oxygen can be fed into the reservoir chamber, said port        comprising a sterilizing filter to prevent contamination of the        reservoir chamber by microorganisms; and    -   7) Port (s) in either the top plate (preferred), outer wall, or        base of the processing chamber through which sensing devices can        be inserted into the processing chamber.

Preferably the general aspect of the system is modified so that itfurther comprises a tubular fluid connector in the processing chamberand disposed, preferably, around the filter element such that saidconnector is not in direct contact with said filter element, saidconnector comprising a sealed end and an entrance end, said sealed enddisposed between the wall of the process chamber and the entrance filterelement, said fluid connector penetrated by the harvest tube, saidsealed end for deflecting fluid flowing from the filter element entranceso that such fluid flows, preferably, through a space separating thefluid connector and the filter element, said fluid connector comprisingan open end through which the deflected fluid can escape into thereservoir chamber.

It is preferred that the general aspect of the system be modified tocomprise a single or plurality of holes in its wall, through which theoxygen is fed into the processing chamber.

It is also preferred that the general aspect of the system be modifiedto comprise additional ports, in the wall of the processing chamber sothat fluid (or fluid containing suspended cells), can be added to thereservoir chamber.

It is also preferred that general aspect of the system be modified tocomprise an an agitation device; exemplified by an open-ended draft tube(open at both ends) inside the processing chamber and surrounding all ora part of the fluid connector tube, said draft tube not directlytouching the fluid connector tube.

In both the preferred and general aspects of the enclosed bioreactorinvention, the line extending from the pump to an external controllerpreferably comprises a sterilizing filter that prevents microorganismsfrom entering the pump. Furthermore, in both aspects, the sealed end ofthe fluid connector tube is modified so that it comprises a poppet valvethe permits fluid to move in one direction, directly from the processingchamber into the space between the valve and the entrance end of thefilter element. However, the poppet valve does not permit fluid to flowin the opposite direction, deflecting fluid flowing from the filterelement entrance so that such fluid flows through a space separating thetube and the filter element.

In both the preferred and general aspects of the enclosed bioreactorinvention, in one preferred embodiment, the system further comprising arigid harvest tube positioned along the center axis of the filterelement, said tube extending from inside the filtrate chamber, throughthe top of the bioreactor, to outside the bioreactor.

In the enclosed bioreactor system, the processing chamber can be used asa bioreactor. As a bioreactor it can be used for culturing assorted celltypes. The filter element (filtrate chamber plus retentate chamber) willfunction as a cell separation device for removal of spent medium andreplacing removed medium with fresh medium. The enclosed bioreactorsystem may be used as a disposable perfusion bioreactor. Such a systemcan greatly simplify the process of continuous culture. It can eliminatethe often complex setup involved in the set up of cell separation systemwith a bioreactor. It can reduce the effort involved in maintaining celllines in continuous culture, as needed in research, development andproduction.

It is desirable that enclosed reactor systems of the invention besterilizable and that, subsequent to sterilization, it can be storedsufficiently sealed to prevent subsequent contamination of its interiorby external microorganisms that otherwise would enter into the interiorof the system subsequent to its sterilization. It is also desirable thatthese systems are in a configuration and made with constructionmaterials that render the system disposable.

The enclosed bioreactor system process, in a general aspect, comprisescirculating fluid back and forth between a processing chamber and aplurality of filtration retentate chambers enclosed within thatprocessing chamber, wherein fluid is driven in alternating directions byvia a pump connected to the filtration retentate chambers, whereinmotion of the fluid through the filtration retentate chambers results intransfer of fluid between the filtration retentate chambers and afiltrate chamber that is separated from the filtration retentatechambers by semi-permeable membranes, said filtrate chamber enclosed bysaid processing chamber.

Normally, material from the filtrate chamber will be harvested at leastonce during the processing of culture.

The processes is preferably carried out using an enclosed bioreactorsystem described herein.

FIGS. 4a-4e show that the many essential features of the enclosedfiltration systems shown in FIGS. 1a-1h are retained. In the case ofFIGS. 4a-4e , however, the processing chamber 211 can be used as a cellculture bioreactor containing a culture of animal cells or othermicroorganisms, where such content serves as retentate 209. As in FIGS.1a-1h , a filter element, 205 is present. processing chamber size,makeup and configuration may be varied according to need. The processingchamber 211 serves the same function here as the process vessel 2, shownin FIG. 1a does. Another similarity with the system in FIGS. 1a-1h is:Fluid connector 203 provides a conduit between filter element 205 andthe processing chamber 211 just as the fluid connector 3 provides aconduit between the reactor chamber 11 and process vessel 2. The fluidconnector 203 directs the flow from the entrance end 242 of filterelement 205 into the reservoir chamber 211. The fluid connectorcomprises a fluid connector entrance and a fluid connector exit. Thefluid connector 203 may be further configured in a manner that directsthe fluid discharging into chamber 211 to maximize mixing within in thechamber, and to increase oxygen transfer into the culture inside thechamber, while minimizing shear. Fluid connector 203 defines aseparation space 284 that permits fluid flow, as shown in one form inFIGS. 4a and 4b , between filter element 205 wall 219 and fluidconnector 203. Further, pump 204 at the base 243 (and 225) of theprocessing chamber 211 is connected to exit end 241 of the filterelement 205, similar to how filter element 5 is connected to pump 4 inFIG. 1a . (Components 6, 7, 8, 23, 27, and 29 of the pump, pump adapter,and pump housing, pump air inlet port in FIG. 1 are similar tocomponents 206, 207, 208, 223, 227, and 229 respectively in FIGS. 4a-4e. Other features shown include, but are not limited to the sterilizingfilter 222 and the line (tube) 221. Another similarity is the fluidreceiving end of fluid connector tube 203 positioned above the filterentrance end 242. A primary dissimilarity between the system shown inFIGS. 1a-1h and that in FIGS. 4a-4e is the extension of the fluidconnector tube 203 from entrance end 242. Where in FIG. 1a , the fluidconnector extends to an external vessel 2, in FIGS. 4a-4e the fluidconnector 203 extends into an internal vessel, reservoir chamber, 211.It extends, preferably, though not exclusively, symmetrically about thefilter element 205, down towards the base 243 of the filter element 205and terminates in the processing chamber above its base 225.

Therefore, during the pump 204 pressure cycle, fluid flows from pumpchamber 207, through the hollow fiber lumens that function as filteringretentate chambers, (FIGS. 4a, 4b, and 4c ; See also 45, 17 in FIGS.1a-1h ). The fluid then exits at the filter element entrance end 242 viaentrances 270 at the entrance ends 271 of those retentate chambers (FIG.4d ). The fluid is then directed into the fluid connector 203 followedby discharge into the processing chamber at the other end of the fluidconnector through opening 230 (See FIG. 4a ). The fluid connector enddisposed between the filter element entrance end 242 and the top plate216 is sealed in FIG. 4a . Therefore, the fluid emerging from the filterelement entrance cannot escape through that end of the fluid connectorbut rather is deflected towards opening 230.

During the exhaust cycle of the diaphragm pump, the direction of fluidflow reverses, flowing from processing chamber 211 through opening 230,into the fluid connector 203, through entrance end 242 into the hollowfibers, out of the fibers via their exits 272 at their exit ends 273,and back into pump chamber 207 (See FIG. 1h for the location of the exitand exit ends of the fibers). In addition to providing tangential flowfor the filtration process, the alternating flow will also providemixing in the processing chamber 211 due to the velocity of fluiddischarging into the reactor chamber 211. FIG. 4 a In addition to themixing generated by the reversible flow of fluid between pump 204 andprocessing chamber, further agitation may be required; an example ofwhich, is shown in FIGS. 4a and 4b ; shown is the presence of a tubularopen-ended draft tube within the processing chamber, to facilitatemixing within the reactor chamber, the draft tube being disposed aroundthe fluid connector 203 but distanced from the fluid connector. Thedraft tube 224 comprises an open draft tube entrance and an open drafttube exit. The air or oxygen bubbled into the processing chamber throughopenings 231, that are positioned symmetrically on the externalperimeter of the draft tube, provide fluid uplift energy, producingcircular flow about both ends of the draft tube; a process wellunderstood in the field and described further later in the text.

In FIG. 1a , the barrier 19 is preferably a selective barrier toregulate exchange between filtrate chamber 10 and reactor chamber 11. Inthe system in FIG. 4a , however, while one may use a selective barrier219, typically, barrier 219 will be a non-permeable barrier in order toprevent the mixing between the adjacent chambers 210 and 211.

Although it is understood that the filtrate chamber 210 may be accessedin a variety of ways or filtered material removed from the system byvarious means, FIGS. 4a, 4d and 4e show an example of filtrate harvestline 213 connected to the preferably rigid filtrate harvest tube 214,the line and tube providing a route for removing filtrate (e.g., cellfree filtrate) from the filtrate chamber 210. Medium removed from theculture as filtrate is replaced with fresh medium for example, viaaddition line 281 connected to one of the available ports (also referredto as “conduits”) connected to the processing chamber. Harvest pump 249controls the rate of filtrate removal. A processing harvest line 244extends from outside the system into processing chamber 211. Line 244optionally can be connected to a manifold of the kind described below orto another line. A pump connected to a harvest line 244 may be used to“bleed” the culture, a procedure commonly used to control cellconcentration. Control of fluid addition or removal to and from thesystem, may be manual or automated using common pumping systems,procedures and controls. Multiple ports and conduits can be inserted inthe wall or top plate of the processing chamber, or be part of amanifold (ports 245, 246, and 247 in FIG. 5a ) connected to lines suchas 244. Others may be used for addition of media, supplements, base,gases or other additives. Other ports may be used as a vent(s) 238 orfor sampling 234 (FIG. 4a ; also manifold in FIG. 5a ). It may bebeneficial to place the harvest tube 214 into the center of the filtratechamber 210, i.e., along the center axis of the filter element 205(FIGS. 4a, 4d and 4e ). Such placement facilitates construction andassembly of the entire system by minimizing the number of walls that theharvest tube needs to penetrate through in the enclosed bioreactorsystem. It also eliminates or minimizes obstructions onto which cellsmay attach and accumulate, as could occur by placement of a harvest tubeperpendicular to the flow path in the fluid connector 203 and processingchamber 211. Filtrate harvest tube 214, especially when rigid, may alsobe used as point of attachment for fluid connector 203 at the pointwhere the tube penetrates through the fluid connector. It may also servefor placement of flow control devices for controlling flow through thefluid connector or other parts of the system, as will be demonstrated.

A system, such as in FIGS. 4a-4e that has to sustain cells at highconcentration and viability must also accommodate culture with itsessential requirements, few examples, of which, are provided, sincethose familiar with cell culture or similar applications know what theessential requirements are:

Oxygen—an adequate oxygen level is a critical component required forsustaining a culture at high cell concentrations. An example of anoxygenation system, is shown in FIGS. 4a, 4b and 4c . Shown is a spargerring 230 discharging gasses into the processing chamber 211 throughpores 231. The sparger ring may be placed symmetrically in the base 225of the reservoir chamber 211 or within pump flange 226. Channel 232,which may effectively extends from the pores to the outer wall 215 ofthe processing chamber is supplied with gasses through channel 232 whichtraverses the processing chamber wall 215 and contains a filter 233 atits inlet to sterilize the inflowing gas. Such a circular tubularsparger ring 230 and sparge pores 231 may be preformed into the pumpflange 226 during its production.

It can be seen that the pores 231 are disposed at the end of theprocessing chamber proximal to the diaphragm pump.

Sparger pores 231 may be positioned along the base 225 to maximizeoxygen transfer, reduce shear and increase agitation. Sparge pores 231or other parts of the air inlet assembly may be equipped with one waycheck valves to assure flow is only in one direction, stopping back flowinto the sparger ring 230. Another method (not shown) for deliveringoxygen into the culture may involve forming a channel in the draft tubesupport frame 252 for delivery of gasses into the draft tube 224 itself.Such draft tube may itself be configured in a manner that oxygenentering the draft tube may be delivered into the culture by a spargemechanism or by a diffusion mechanism by methods familiar to those inthe field.

Agitation—Mixing of the culture, also a critical aspect of a suspensionculture, may be provided by a number of known mechanisms; one example,shown in FIGS. 4a-4b , where draft tube 224, is placed centrallysurrounding the filter element 205. In combination with the spargerpores 231, the updraft created by the rising bubbles generate an upliftof fluid flow within the draft tube. A fluid updraft may be created bybubbles on the outside of the draft tube 224 and a fluid return indowndraft through the center of the draft tube exiting opening 229,between the base of the draft tube and the reactor base 225, to resumethe circular flow. Note that diaphragm pump flange 226 and the reactorbase 225 may be shaped to minimize formation of dead zones for cellaccumulation and to facilitate circular flow of the culture. The top endof the draft tube is maintained below liquid level 237 when there isliquid in the reservoir. This “air-lift” method of agitation is a wellunderstood process and may be varied by those familiar with the process.Attachment and positioning of the draft tube may be accomplished invarious ways. As shown in FIG. 4a , it may be secured to the reactorwall 215 through draft tube support frame 252, or to the top plate 216,or base 225 by other attachments. A vent tube 238 at the top of thereactor will provide a vent for the added gas or for gas flow ingeneral.

Another possible form of agitation involves taking advantage of thealternating flow caused by the diaphragm pump. As shown in FIGS. 4d and4e , It is possible to incorporate a one directional check valveassembly 225 into the fluid connector 203, which may direct flow throughthe fluid connector and flow direction in the processing chamber 211. Asillustrated in FIGS. 4a and 4d : During the exhaust cycle of the pump204, overall flow is from the processing chamber 211 to the pump chamber207. At least part of the flow is directed through poppet valve opening246 and part of the flow will proceed through opening 230, as previouslydescribed. Poppet valve 228 (See FIGS. 4d and 4e ) which may be aflexible material, will be forced away from opening 246 by the negativepressure generated within the fluid connector relative to the reactorchamber and the resulting force generated by the flow of fluid flowingfrom the processing chamber 211 through port 246, into the filterelement 205 on its way to pump chamber 207, (See FIG. 4a .) During thepump pressure cycle, on the other hand, when flow direction reverses,the flow emerging from pump chamber 207 flows into the filter element205, emerging at the entrance end 242 and forcing puppet valve 228against opening 246, effectively blocking further fluid flow throughthose openings; thereby, flow can only proceed through the fluidconnector 203, emerging from opening 230. (See FIG. 4a .) The processoffers circular flow within the processing chamber 211 to facilitatemixing. Orientation of the valve 225 or its configuration may be variedaccording to need. Other common forms of agitation may be used tofacilitate mixing within the processing chamber.

Temperature control for the system may be accomplished in a variety ofways including using a thermal blanket, water or air jacket a heatingelement, etc.

The enclosed bioreactor system system described can be customized forvarious uses and to achieve optimal results.

Another example of the enclosed bioreactor system is shown in FIG. 4f .The example in FIG. 4f shows an enclosed bioreactor system where theprocessing chamber 211 is inside a bag 215 and which also serves as anenclosure for the entire system, analogous to the system in FIG. 4a .The filter element 205 placement, however, is horizontal in thisexample. Also the draft tube 224, and the air sparging pores 231 presentin FIG. 4 are absent here, other forms of agitation and oxygen deliveryto the culture are provided. The pump 204 is connected to the filterelement at one end, the exit end 243; at its other end or entrance end242, the filter element is connected to the fluid connector 203 or theentrance end may discharge directly into the bag. As previouslydescribed, fluid flow generated by the diaphragm pump flows reversiblybetween pump chamber 207 and processing chamber 211 through the filterelement 205. Filtered harvest may be collected from filtrate chamber 210using port 212 and harvest line 213 and pump 204; further additions orsubtractions from the system may be accomplished through other lines246, 247, or others if necessary. Additionally, in FIG. 4f , components54, 206, 208, and 219, are essentially the same or functionally the sameas components 54, 206, 208, and 219 in FIGS. 4a, 4b, 4d and 4e . It isevident that there are other components in FIG. 4f that, although notnumbered, have the same meaning as FIGS. 4a, 4b, 4c and/or 4 d.

Accordingly, the fluid filtration system in FIG. 4f can be considered tobe a simplified version of the fluid filtration system in FIG. 4a .Absent in FIG. 4f are connector tube 203 encircling the filter element205, a cylindrical draft tube 224, sparger ring 230 and pores 231 foroxygen bubble entry.

The examples provided are to demonstrate some, but not all of thepossible configurations of the system. One can envision a system wherethe pump is in the upper position and the filter element is below thepump. Fluid ejected from the diaphragm pump flows into the top of filterelement and ejected into the reactor camber at the lower end of thefilter. Such an embodiment is illustrated in FIG. 4g . (Note that inFIG. 4g , post 250 and the port for line 244 are only visible below thetop plate 216. Those ports extend above top plate 216 but the portionsof those ports above the top plate are not visible in FIG. 4g becausecylindrical component 226, which comprises part of the pump flange,obscures them from view for purposes of that Figure.

One can further envision a system where the diaphragm pump is notconnected to the filter element directly but through a conduit. Otherexamples are also possible.

Sampling Manifold and Process

The manifold invention in a general aspect comprises;

-   -   (1) a channel (such as the internal channel of a tube), the        channel comprising a first end and a second end;    -   (2) an alternating flow diaphragm pump connected to the first        end of said channel and tube, and    -   (3) a plurality (more than one) of probe ports located on the        channel at a positions between the two ends of the channel;        wherein the second end of the channel is connectable to a fluid        source (such as a vessel or reactor chamber).

Ports provide places where a probe or sensor may be inserted forpurposes of sampling or monitoring the fluid in the channel. Ports alsoprovide places where, additions or subtractions to the content in thechannel can be made. When used for sampling, each probe port isconnected to a probe device. Each probe device will be a device thatmeasures a physical or chemical property of fluid within the channel.The measurements can include, but are not limited to, measurement ofpressure, pH, or the concentration of a particular material present inthe fluid.

The manifold invention may further comprise a filter element within itschannel and a filtrate chamber disposed between the filter element andone or more ports. The filter element will be, for example, a hollowfiber filter cartridge. The outer wall of the filter cartridge ispreferably fully permeable so that the size selection step for smallersubstances is controlled by the semi-permeable membrane walls of thehollow fibers.

The manifold sampling process of the invention comprises:

-   -   (1) causing fluid from a container (or chamber or compartment)        to enter a manifold channel;    -   (2) and then causing the fluid to mostly or entirely exit said        channel so as to return to the container, wherein the motion of        the fluid controlled by an alternating flow diaphragm pump, and    -   (3) measuring a property of said fluid while it is in said        channel, said measurement accomplished by probe device connected        to said channel, said probe device capable of measuring a        physical or chemical property of said fluid.

The process is preferably done using a manifold system described above.

Rapid fluid equilibration between the filtrate and retentatecompartments (or chambers) in an alternating tangential flow process asdescribed herein can facilitate such measurements such that one maysample the retentate and filtrate in the same sample stream.

FIGS. 5a and 5b illustrate two embodiments of the sampling manifold, 259and 260. The connection 244 from the manifold to a process vesselfacilitates severing the manifold from the bioreactor or its connectionto the bioreactor; and facilitating the performance of such proceduresin a sterile manner. The disposable nature of a bioreactor system (e.g.,FIGS. 4a and 4f ) is enhanced by the use of a removable samplingmanifold, which via line 244 in FIG. 4a could, for example be reversiblyconnected to a bioreactor While it is possible to insert probes directlyinto processing chamber 211 (FIG. 4a ), it would not be convenient to doso. Individual probes can be expensive; and if supplied with thebioreactor, greatly add to the cost of the system. Direct insertion ofprobes into a presterilized bioreactor risks contamination; such probescan fail in mid run or drift out of calibration rendering them uselessand therefore jeopardizing the culture. Probes can be large, not easilyaccommodated by a small culture system, as may be necessary. It would bebeneficial therefore to incorporate a device such as the presentmanifold invention capable of a single sterile connection, frequentlysampling the culture and monitoring the status of the culture byanalyzing the samples. Such sampling would have to be sufficiently rapidto monitor rapid changes in the culture. The manifolds 259 and 260 shownin FIGS. 5a and 5b , are capable of holding multiple and varied probessuch as 261, 263, 264, and 265. The outputs from the probes may beconnected, for example via a line 262, to a controller device or devices(not shown) capable of monitoring such outputs and provide data outputand control capability of culture parameters such as pH, DO, CO2, andothers.

Manifolds 259 and 260 both contain a channel (e.g., a tube), 266 and 279respectively, which at one end (a first end) is connected to the pump274 and at the other end (a second end) is connectable, for example, toa bioreactor, bioreactor system, or filtration system, through line 244so as to allow fluid to flow between the manifold and the bioreactor.Channels 266 and 279 are connected to a diaphragm pump 274. Such pump issimilar to the diaphragm pumps 4 and 204 and has two chambers, 277 and278 separated by a diaphragm 276. The pump 274 is capable of receivingand expelling fluid through pump chamber 277 and process chamber 211(See FIG. 4a ), as previously described for pumps 7 and 207. Thefrequency of fluid cycling between vessel and pump, through the line 244and probe manifold can be controlled by controlling the cyclingfrequency of diaphragm pump 274. This provides a convenient method forcontrolling the culture sampling rate. Probes 261, 263, 264, and 265 canbe secured in the manifold 260 with their sensing ends exposed into themanifold channel 266 or 279. Fluid within the manifold channel 266 or279 can thus be probed and monitored. The probe placement within themanifold must be in a manner to prevent contamination of the fluidwithin the manifold from an external sources and to confine the fluidwithin the manifold from escaping. Although less preferable, it is notbeyond the scope of the concept being described to use a continuouspump, such as a peristaltic pump, to remove culture media through oneconduit, directing the sample through a manifold 260 for analysis, thanreturn the sample to the culture vessel through another conduit.

There are other substantial benefits to the described sampling manifold260. The following are some examples:

1. The sampling manifold may also incorporate multiple ports, such as267, for making additions and subtractions to and from the culture inthe bioreactor, for example via a line 234, 245 or 247, and for example,pump 248. Thereby, the number of ports that need to be added to theenclosed reactor system can be greatly reduced, facilitating thatsystem's construction and simplifying its use.

2. A single connecting line 244 between sampling manifold 260 and anenclosed reactor system such as 201 shown in FIG. 4a , allows quickattachment or detachment of the sampling manifold to the vessel using acommon tube welder or similar sterile connectors. Should a probe fail orrequire calibration, one sampling manifold can be rapidly exchanged withanother, greatly reducing the risk to the culture.

3. Such sampling manifolds may be prepared and sterilized separatelyfrom a bioreactor, such as 201 shown in FIG. 4a , greatly adding to theconvenience of their use, and sterilization.

4. The sampling manifold may be readily used with disposable bags. Oneof the limitations of disposable bags is the difficulty in placingmultiple probes into the bag and in monitoring and control of cultureconditions within the bag. The probe manifold such as described canalleviate this handicap.

5. The described manifold may be modified in a manner, as shown in FIG.5a that includes a filter element 285 (such as a hollow fiber filtercartridge) in channel 266. When the filter element 285 is used, it ispreferred to use a manifold filtrate chamber (or compartment) 286surrounding the filter element and in fluid contact with the ports 263,264, and 267. The position of the filter element 285 in FIG. 5a is a oneposition; however, the filter element and filtrate chamber 286 can besimilarly positioned elsewhere inside channel 266. Culture media willflow reversibly between chamber 211, containing the culture, and pumpchamber 277.

6. The ability to remove culture samples from the bioreactor offers theuser with additional diagnostic capabilities not readily possible withfixed probes within the bioreactor; for example, the flow of a sample,flowing from the bioreactor to the pump can be stopped for a certainduration, during which the decay rate of oxygen concentration can bemonitored, reflecting the condition of the culture; similarly, the rateof change in other culture parameters, glucose, CO2, pH, and others, maybe repeatedly monitored without disturbing the culture. Repeated removalof samples from the bioreactor is not required, thereby reducing therisk of contamination and change in the sample.

7. The ability to form a filtered stream by filter element 285 allowsdirecting of the stream by a filtrate line 247 to a secondary analyticaldevice such as a HPLC or some other analyzer.

The filter element 285 can be held in position within channel 266 by two0-rings 258. However, the filter can also be positioned and sealedwithin channel 266 using adhesives or mechanically.

A manifold filtrate chamber 286 formed between the filter and the wallof channel 266 may be probed by the probes exposed to channel 266.Preferably the filter element 285 has a fully permeable outer wall. Thealternating flow between chamber 211 and 277 facilitates flux of fluidsbetween the retentate and filtrate compartments in manifold 260. (Theretentate compartment of the manifold is, for example, the spaces insidethe hollow fibers of the filter element 285 plus the portion of channel266 not occupied by the filter element).

The alternating flow through the manifold between bioreactor and pump274 enables the probe manifold to accurately reflect the condition of aculture. One can extend this concept to include monitoring of cultureconditions either in the retentate, filtrate, or processing chambers ofa system, and in parallel streams if desired. Monitoring the conditionof the culture in a filtered stream, free of cell debris, can extend thelife of the probe by minimizing accumulation of debris on the probes;the filtered stream may also be directed to other analyticalinstrumentation that require a filtered sample. This provides the userwith the ability to monitor cell growth and culture activity on anongoing basis. Such a device is illustrated in FIGS. 5a and 5 b.

Dual Pump System

The Dual Pump Invention

Another invention is a dual pump system, said system comprising:

-   -   1) a first pump, said first pump being a two-chambered diaphragm        pump, said first pump comprising a first chamber that is a pump        reservoir chamber connectable to a source of fluid, said first        pump further comprising a second chamber being an interface        chamber connectable to a conduit, said first and second chambers        being separated by an elastically deformable diaphragm;    -   2) a conduit connected to the interface chamber of said first        pump; and    -   3) a second pump, said second pump comprising an interface        chamber connected to said conduit, said second pump interface        chamber comprising a movable element selected from the group        consisting of an elastically deformable diaphragm or a        non-elastic, piston-like, movable wall, said second pump being        connectable to a pressure controlling mechanism.

In one embodiment, said pressure controlling mechanism is connected tothe second pump exclusively (not also to the first pump). In anotherembodiment, said pressure controlling mechanism is connected to both thefirst pump and the second pump, for example by using a peristaltic pump.

Examples of the source of fluid include but are not limited tobioreactor chambers, for example, the retentate chamber of the enclosedfluid filtration system or enclosed bioreactor systems described above

In one embodiment, the second pump is also an alternating flow diaphragmpump, such that the pump comprises an elastically deformable diaphragm.In another embodiment, the second pump is a mechanical pump, such thatthe pump comprises a movable wall, whose reversible movement and pumpingaction are imparted by well established methods. One example of manypossible mechanical pumps is one where the movable wall is a piston (apiston wall) that is part of a piston pump driven by a cam mechanismcoupled to a motor or a step motor. It is well understood, thereversible piston movement is determined by the cam stroke In anotherembodiment of a possible mechanical pump, the movable wall comprisescoupling, directly or indirectly, to a screw or threaded drive shaftwhich in turn is coupled to a motor or stepper motor. Rotation of thedrive shaft causes, in screw-like fashion, the wall to move. Thedirection of shaft rotation (and therefore the direction of wallmovement), and the distance traveled by the wall, can be controlled bydirection and duration of rotation of an electro-mechanical rotarydevice and/or by configuration of the screw shaft. The rate of pistonmovement in either of the above two embodiments may be controlled by therate of motor rotation, by the pitch and pitch direction of the screw;it is also understood that the second pump chamber and wall must besealed and leak proof in order to transfer all its energy to the firstpump.

It is preferred that the interface chamber of the first pump be largerthan any other chamber of a pump in the two pump system, thus limitingthe travel of the diaphragm within the first diaphragm pump.

Preferably the interface chamber of the first pump, the interfacechamber of the second pump, and the conduit connecting them, arepreferably filled with a non compressible medium, preferably a liquid.

Optionally, the dual pump system further comprises sensors in one ormore chambers, especially the interface chamber of the second diaphragmpump, for added control of pump action. Also, optionally, one or moreadditional pumps are connected to the two pumps of the dual pump systemthrough air, liquid or mechanical coupling capable of controlling theaction of the pump(s) in the multiple pump system.

The dual pump pumping process comprises cycling a first and second pumpthrough one or more pump cycles, the pumps being connected to each otherby their respective interface chambers, the first pump being atwo-chambered diaphragm pump which, in addition to its interfacechamber, comprises a pump reservoir chamber connected to an externalsource of fluid, wherein the interface chamber of the second pump isexposed to a pressure emanating from an external source of pressure (apressure controlling mechanism) that is alternately less than or greaterthan the pressure in the external source of fluid.

Using a single pump system (e.g., rather than a double pump system),driven by air pressure (For example in FIGS. 1a and 4a ) has provenquite effective, particularly at large scale. However, it suffers from anumber of features that negatively effect its reliability and pumpingaccuracy. Such shortcomings are derived from the inherentcompressibility of air. For example, referring to FIGS. 1a and 1b duringthe pressure cycle, gas (usually air) flows into the pump's firstchamber 8, pressurizing and compressing the gas therein. Having aflexible diaphragm 6 between the pump's air first chamber 8 and itssecond chamber 7 separates the content in the chambers and permitsexpansion of first chamber 8 during its pressurization, simultaneouslydriving the liquid from the second chamber 7 towards process vessel 2.The resulting flow rate is a function of the pressure difference, b.P,between the first chamber 8 and process vessel 2. For the purpose ofdescription, assuming the pressure in vessel 2 remains constant, thepressure in chamber 8 required to drive diaphragm 6 at a specific rateis term “driving pressure”; therefore, with increasing b.P, and theresulting increase in driving pressures, hence, flow rates, it becomesmore difficult to control diaphragm position. Slight changes in serviceair pressure, inherent delays in response of sensors, regulators, valvesor controller electronics can effect the final position of the diaphragmat the end of the pressure cycle.

Similar difficulties arise during the exhaust cycle. As gas pressure inpump first interior chamber 8 is reduced relative to process vessel 2,flow direction reverses from vessel 2 towards the diaphragm pump 4. Therate of flow will depend on the LiP between the vessel 2 and diaphragmpump 4, and is termed “negative driving pressure”.

However because of the compressibility of gases, generating similar +LiPor −LiP during the pressure and exhaust cycle, respectively, does notresult in similar flows in both directions. To determine the flow ratesto and from chamber 8, one may use the following equation:Flow rate=chamber 8 displacement volume/displacement time,It is apparent that by maintaining same flow rates to and from chamber8, the duration of the respective pressure and exhaust cycle will not besimilar; pressurizing chamber 8 relative to vessel 2 compresses the gaswithin that chamber; thereby, increasing the mass of the gas within thechamber; conversely, transitioning to the exhaust cycle decreases thepressure in chamber 8; effectively requiring longer to clear the gaseousmass from chamber 8. Efforts to establish constant cycle periods duringpressurization and exhaust can be achieved but at dissimilar gas flowrates, an undesirable effect.

Another effect of using a gas to drive a single pump alternatingtangential flow system is observed during cycle transitions.Transitioning to a pressure cycle, the pressure in first interiorchamber 8 needs to switch from full exhaust to pressure drivingpressure; similarly, transitioning to the exhaust cycle, the pressure inchamber 8 needs to switch from full pressure to negative drivingpressure; such drastic changes in pressure during an alternating flowcycle result in a short delay during cycle transition, and are referredto as a “soft transition. Such a delays becomes severe at higher flowrates, where fluid flow momentum becomes increasingly significant indisrupting the transition. These are some factors that can greatlycomplicate cycle flow control accuracy. While a soft transition may bebeneficial in many applications, it may not be beneficial in manyothers. It is well known for example that any reduction in tangentialflow, (technically reverting to the less efficient “dead endfiltration”), during the soft transition can decreases the life of thefilter or reduce its filtration capacity. Uninterrupted or undelayedcycle transition may be more desirable, particularly where a moreconsistent alternating tangential flow is essential; such undelayedcycle transitions are termed “hard transitions”,

Yet another potential problem of using one diaphragm pump is inherent inthe use of a single diaphragm to separate the pump into an air drivechamber and a liquid chamber. Should the diaphragm rupture, pressurizedair will flow into the process vessel unrestricted, creating potentiallya hazardous condition. Also liquid may flow towards the controller,potentially contaminating the process and damaging the controller.

It is possible to compensate for some of the described shortfalls, ofthe air driven single pump system, with various control schemes sensors,pneumatic devices, process modifications, or other possible schemes.Nevertheless, it would be more desirable if diaphragm cycle or flow ratecould be controlled with greater precision and reliability during thealternating flow cycles. It would also be preferable if the integrity ofan enclosed reactor system remain intact and uncontaminated should adiaphragm rupture. In some cases, it would be highly desirable ifsources of pumping energy other than compressed air or vacuum are usedto drive the pump, particularly in those cases where compressed air orvacuum services are limited or not available. FIGS. 3a, 3b, 3c and 3 eprovide examples of methods, other than the direct air driven singlepump system described.

FIG. 3a shows an enclosed filtration system 101 that comprises a dualpump system which is an improvement over using only the diaphragm pump104.

It can be seen however, that many features of the enclosed filtrationsystem 1 as described in FIGS. 1a-1h are present in system 101 shown inFIG. 3a . For example, components 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, and14, in FIG. 1a are present as components 103, 104, 105, 106, 107, 108,109, 111, 112, 113, and 114, respectively. Further examples arecomponents 15, 16, 19, 24, 25, 29, 40, 41, 42, and 43, in FIG. 1a whichare present as components 115 (albeit sufficiently long to include thesecond pump 144), 116 (although it needs cross hatching becausesectional view), 119, 124, 125, 129, 140, 141, 142, and 143,respectively. All of the foregoing components common to FIGS. 1a and 3aare also present in FIG. 3 b.

In FIG. 3a , however, a second pump 144 is incorporated, to form a dualpump system, the two pumps connected in series through conduit 135.Conduit 135 may be short, minimizing the length of the connectionbetween the two pumps as in FIG. 3a ; this allows combining the entireenclosed reactor system with both pumps, to be combined into one module.Conduit 135 may be long, allowing separation of the two pumps as in FIG.3b . Either choice of conduit allows greater flexibility as to thechoice of method of driving the pump, including but not limited to theuse of air, motor drives or other mechanical devices.

In FIGS. 3a and 3b , the examples of the dual pump system show the useof a second pump 144 that is similar to the first pump 104, i.e., isalso a two-chambered diaphragm pump. Like first pump 104, second pump144 contains two chambers 137 and 138 separated by a diaphragm 136.Chamber 107 (also referred to herein as the “pump reservoir chamber”) inthe first pump 104 remains the same as previously described. Itcontinues as a reservoir for receiving or expelling retentate flowingfrom or to a vessel (see for example vessel 102 in FIG. 1a ); however,unlike the air driven system previously described, chamber 108 (alsoreferred to herein as the “interface chamber of the first pump”) in thefirst pump 104 connects with chamber 137 (also referred to as the“interface chamber of the second pump) of the second pump 144 throughconduit 135. In addition, chamber 108 of the first pump 104, chamber 137of the second pump as well as conduit 135 are preferably filled with anon compressible medium, preferably a liquid (The 139 refers to thelocation of the liquid when such an embodiment is used.) The secondchamber 138 of the second pump 144 may drive the alternating pump 104cycle, by using air flow to or from chamber 138 or by changing thevolume of chamber 137 by some other mechanism as described. The secondchamber 138 of pump 144 may be connected, optionally, via a sterilefilter 122 and a line 121 to an air pressure controlling mechanism ordevice 154 that can exert a positive or negative pressure on thecontents of that second chamber. In the case where gas is used, adding agas to chamber 138 will drive the diaphragm 136 and cause the chamber toexpand. This in turn will drive the liquid in chamber 137 outwardthrough conduit 135 into chamber 108, expanding that chamber. As before,expansion of chamber 108 will drive the retentate in chamber 107 towardsvessel 102, provided that the pressure in chamber 108 is higher than invessel 102. Conversely, exhausting chamber 138 and reducing its pressurerelative to vessel 102, will cause non compressible liquid at location139 to flow from the first pump chamber 108 to the second pump chamber137; in turn, retentate flows into the first pump chamber 107 fromvessel 102. The following are some benefits of the described double pumpsystem:

One benefit is that non-compressible liquid at location 139 may bevaried depending on the application; for example, when used in a cellculture system, a non-compressible liquid at location 139 may be PBS(phosphate-buffered saline) or the culture medium itself. As furtherprotection, non-compressible liquid at position 139 may be sterilizedsimultaneously with the entire enclosed reactor system or added into thepump chambers following sterilization in a sterile manner through adedicated port (not shown). Therefore, should diaphragm 106 rupture, thenon-compressible liquid at location 139 will spill into the retentatepathway 109 without harming the culture. Should either diaphragm 106 or136 rupture separately, the culture will remain protected against totalloss and may, in fact, be continued until the run is completed under a“safe” mode or allow the exchange of the damaged enclosed reactor systemwith a new enclosed reactor system.

Another benefit of the dual pump system shown in FIG. 3a involves theuse of different size of chambers 107 and 137; for example, volume ofchamber 107 can be made larger than chambers 108 and 137. This becomesuseful if expansion of diaphragm 106 into chamber 107 has to limited,e.g., to prevent its extension to the wall of the chamber. Smallermaximum volumes of chambers 108 and 137, using an inelastic fluidcoupling in between will displace a volume smaller than the maximumvolume of chamber 107. Chamber 107 will remain unfilled by thedifference in volumes Where previously, if chamber 108 was overpressurized, as was common at high flow rates, it was somewhat difficultto control diaphragm 106 position; the diaphragm would be commonlyforced to extend to the walls of chamber 107 with potentially harmfuleffects on the cultured cells. In the modified system a second pump 144with smaller chambers 137 would eliminate such over extension ofdiaphragm 106.

Some other benefits of the dual pump system involve added flexibility incontrolling alternating tangential flow. In FIG. 3a , for example,where, the conduit 135 between the two pumps is inelastic, it providesthe means of controlling the first pump 104 with a “remote” second pump144. It becomes possible to incorporate sensors 146 and 147 into thesecond pump 144 for locating the position of the diaphragm within. Thatinformation may than be used for precise location and control of thediaphragm in the first pump 104. Where, with the use of a single pump,which has to remain sterile, the use or insertion of sensors is greatlylimited. The use of a second pump removes that limitation. Positioningdevices or sensors based on proximity, pressure, contact and opticalamongst many others that may be incorporated preferably into the secondpump without affecting the first pump or the culture within and withminimal effect on the process; this may be achieved before or aftersterilization of the system since the two pumps are separated by animpermeable diaphragm. In addition, the use of the second pump opens thepossibility of using sensitive sensors or positioning devices thatotherwise may be damaged by the sterilization process; in such cases,the first pump could be sterilized by usual means, while the second pumpcould receive labile, unsterile sensors. Placement of sensors into thesecond pump is less critical since, as indicated, the pumps areseparated by an impermeable barrier. By retaining the liquid couplingprovided by the liquid at location 139 between the two pumps, it becomespossible to include agents in the liquid that could neutralize potentialcontaminants. The proposed system would protect the culture fromcontamination while retaining a high degree of pump control.

Another benefit of the dual pump system, as indicated, involves theability to couple the enclosed reactor systems to energy sources otherthan compressed air or vacuum. One may couple the first pump 104 to asecond diaphragm pump 144 through a liquid coupling as to a second pump144 where the second pump drive system is provided by an electric motoras shown in FIGS. 3b and 3 c.

FIG. 3b shows second pump 178 connected via an electric (hereperistaltic) pump 152 to first pump 104. The liquid flow between thesecond pump chamber 176 and the first pump chamber 108 is generated byan electric pump 152 (for example, a reversible peristaltic pump),through conduit 171, for example tubing, which connects to the interfacechambers of both pumps. The peristaltic pump 152 would pump the liquidfrom chamber 176 to chamber 108 in one direction, “pressure” cycle. Whenthe liquid in chamber 176 has been pumped out, a sensor 179 on chamber176 would signal for change in pumping direction. Similarly, sensor 180would signal the end of the “exhaust” cycle. Any number of pumpingmechanisms may replace a peristaltic pump. Numbers in FIG. 3b that alsoappear in FIG. 3a represent many, but not all, components common to thesystems in the two figures.

Other components of the system in FIG. 3b are first pump connector tube170, second pump connector tube 172, second pump housing components 173and 174, second pump diaphragm 175, the first chamber (interfacechamber) 176 of the second pump, second chamber 177 of the second pump

FIGS. 3c and 3d show a variation of the dual pump system of shown inFIG. 3a . FIGS. 3e and 3f show another variation of the dual pump systemof shown in FIG. 3a . The second pumps in FIGS. 3c-3d and theirstructures are well known in the art.

In FIGS. 3c and 3d , the second pump 644 is a piston pump. There isliquid coupling 621 (e.g. tubing) between the two pumps of the dual pumpsystem. (A cam mechanism, shown in FIGS. 3c and 3d in two differentconfigurations (on the left and right of the Insert respectively) with638, 630 and 634 numbering its component parts in its twoconfigurations, is connected to the motor drive shaft 630 and may beused to generate an eccentric stroke via lobe 634, which when coupled topiston 636 of the second pump 644 (also referred to as the piston wallof the second pump), may be used to generate the exhaust and pressurestrokes. The pressure cycle will be generated as the cam 638 moves thepiston 636 into pump chamber 637, expelling the contents in that chambertowards chamber 108 in the first pump 104. The exhaust cycle isgenerated by the continued rotation of the cam mechanism and withdrawalof piston 636 from chamber 637; in the process, chamber 637 expandsreceiving fluid flowing from chamber 108. The volume of chamber 637 maybe set to equal the volume of chamber 108, thus setting the diaphragmdisplacement stroke of the first pump 104; furthermore, fluid flow ratecould then be accurately controlled by controlling the electric pumprotational speed.

FIGS. 3e and 3f demonstrate another example of the second pump 656 beinga piston pump here driven by a reversible screw drive 658. Thereversible screw drive is coupled to a motor drive shaft 652. The linearmotion of piston 653 (also referred to as the piston wall of the secondpump) within pump housing 657 is than be established by the motorrotation rate, size of the pump chamber 654 and the pitch and length ofthe reversible screw 658. The threaded screw 658 may be coupled topiston 653 directly or indirectly; in the first case, the screw may becoupled to the piston by passing through threaded opening 659 in thepiston 653, so that rotation of the screw causes the piston to move; inthe second case, the piston is added to an unthreaded part of the screwshaft 658, not shown. A separate, threaded coupling is placed on thethreaded portion of the shaft like in the first case, and like the firstcase rotation of the shaft causes the threaded coupling to move. A unionbetween the threaded coupling and piston imparts the movement of thethreaded coupling on to the piston. The indirect coupling is preferableby simplifying sealing between surfaces. Where sealing a threadedcoupling can be complicated, sealing a round tubular object, pistonopening 659, against a round shaft, part of the reversible screw 658,using an “O” ring, a gasket, mechanical coupling, etc. is much simplerand reliable. Similarly, the peripheral diameter of the round piston canbe sealed against the round inner pump chamber wall 657. It isunderstood that pump chamber 654 in FIGS. 3e and 3f and chamber 637 inFIGS. 3c and 3d must be sealed in order to transmit their energy throughconduit 651 to the first pump chamber 108. Elimination of leaks allowsaccurate control of liquid displacement or flow control to and fromchamber 637 and chamber 108. There is a liquid conduit 651 (e.g. tubing)for the two pumps of the dual pump system.

The second pump e.g., pump 144, need not assume a specific shape or becomposed of specific materials as long as it serves as means forreversibly pumping liquid into chamber 108 of the first pump 104.

Note that in FIGS. 3c-3d and FIGS. 3e-3f , 636 and 653, respectively,are movable pistons, the center hash representing an “O” ring or someother gasket. Chambers 637 and 654 are similar to chamber 7 in FIG. 1a .Liquid in those chambers will flow from those chambers to the first pumpchamber 8, to drive the diaphragm in the first pump. In FIGS. 3e-3f ,658 is a reversible screw that drives piston 653 reversibly.

By their nature, the cam or reversible screw set the stroke of thepiston; so as the motor is turning, the piston moves back and forth by aset distance. With an air driven diaphragm pump or reversibleperistaltic pump as in FIGS. 3a and 3b , the controller needs to knowwhen the stroke ends, therefore, one uses a device such as a sensor toinform the controller of when the cycle ends and its time to reversedirection.

Any numbers in FIGS. 3c, 3d, 3e and/or 3 f that also appear in FIG. 3arepresent components common to the systems in which they appear. Allcommon components not marked by numbers are nevertheless recognizable ascommon components.

The pump descriptions herein do not limit the number of pumps that maybe used in series or parallel with the first pump 104. For example athird pump may be used to drive a second diaphragm pump by anon-compressible coupling. The second diaphragm pump may than be coupledto the first pump, also, through a non-compressible coupling. The thirdpump may be a piston or diaphragm pump and driven by an electric motoror some other means; for example, chamber 138 of second diaphragm pump144 in FIG. 3a could be coupled to the third pump's piston or adiaphragm chamber through a liquid conduit, as previously described.Chamber 138 would cycle in accordance with the cycling of piston ordiaphragm of third pump. The second pump 144 may than serve to drive thefirst pump 104, through a liquid coupling, as previously described. Theadvantage of such a system is the ability to fully sterilize the firstand second diaphragm pumps as well as the liquid connection between themand yet maintain a none sterile liquid connection between the second andthe third pump. In this manner, one can retains a sterile environmentshould either diaphragm in the first pump or the second pump rupture andat the same time retain the pumping accuracy and control offered by anelectric pump. Such a system could offer the high level of reliabilityrequire in dialysis applications, or some other medical or critical,none medical, application.

The Modifier Module Invention

The invention is a modifier module designed for use inside filtrationand bioreactor systems so as to modify some (or less commonly, all) ofthe components in the system.

The modifier module (preferably columnar in shape), comprises in onegeneral aspect:

1) a scaffold; and

2) a population of modifier agents bound to said scaffold.

Optionally, the module further comprises a semi-permeable membrane thatsurrounds the population of modifier agents and, in conjunction with thescaffold, encloses that population.

The modifier module (preferably columnar in shape), comprises in anothergeneral aspect:

-   -   1) a scaffold;    -   2) a semi-permeable membrane partially or completely surrounding        said scaffold in a manner that allows a compartment between said        membrane and said scaffold; and    -   3) a population of modifier agents in said compartment; wherein        the semipermeable membrane is not permeable to the agents but        permeable to molecules small enough to pass through the        membrane; and wherein the modifier agent population is retained        within the compartment (preferably stacked against the        scaffold).

Examples of modifier agents are antibodies or enzymes.

A modifier agent population bound to the scaffold can coat the surfaceof the scaffold (e.g., where the agent is a resin).

A modifier agent population may be part of or attached to beads,particularly where it is not bound to the scaffold but rather held inposition within the membrane-scaffold compartment.

The modifier agent is preferably selected from the group consisting ofan antibody, an enzyme, a non-enzymatic catalyst, a receptor, a ligand,a chemical that will modify a biological molecule, an affinity resin,and an ion exchange resin, a biological receptor, a ligand that willbind to a biological receptor, and a chemical that will modify abiological molecule.

Of particular interest are modifying agents that can bind or modifycomponents, such as those that may accumulate in the kidneys and bloodto undesirable levels and can be removed using a filtration system, suchas the enclosed filtration system described herein. Components thatcould be considered undesirable include, but are not limited to, toxinsgenerally, inflammatory proteins (such as plasma C-reactive protein(CRP) and amyloid A (SAA)), colony stimulating or growth factors,chemokines, (such as a member of the leukocyte chemoattractivecytokines, also known as CXC, CC, C and CX3C chemokines),pro-inflammatory interleukins (for example, IL-1, IL-6), tumor necrosisfactor-c,: (TNF-o:)], pancreatic secretory trypsin inhibitor (PSTI), HDLcholesterol (HDL), low-density-lipoprotein (LDL) cholesterol, hormones,urea, salts, drugs, and vitamins.

A modification process of the invention comprises the steps of:

-   -   (1) contacting a fluid with a modifier module of the invention,        and    -   (2) filtering the fluid using a semi-permeable membrane, wherein        the modifier module is within either a chamber of a filtration        system or a chamber of a bioreactor system, preferably an        enclosed filtration system or enclosed bioreactor system of the        invention.

With the enclosed filtrations systems of the invention described herein,the preferred location of the modifier is the reactor chamber, and thepreferred sequence is that step (2) follows step (1). In one embodiment,the process further comprises a step (3) where the filtered and modifiedfluid is administered to a human, especially either via dialysis, byinjection, or orally.

Modification of Reactor Chamber Fluid and Other Fluids

A potential application of a modifier module 351 is shown in FIG. 6a .In that Figure, the reactor chamber 311 is designed to accept a modifiermodule 350, whose function is primarily to affect the composition offluid in that chamber. Accordingly FIG. 6a illustrates an embodiment ofthe enclosed filtration system of the invention.

Many features of the enclosed reactor system 301 in FIG. 6a are presentin system 1 of FIGS. 1a, 1b, 1c, 1d, 1e and 1f . Components 303, 306,307, 308, 309,312,313,314,315,316,321,322,323,324,325,326,327,329,335,340,341,342, and343, are the same as components 3, 6, 7, 8, 9, 12, 13, 14, 15, 16, 21,22, 23, 24, 25, 26, 27, 29, 35, 40, 41, 42, and 43 respectively, inFIGS. 1a, 1b, 1c, 1d, 1e and 1f . Additional marked components in FIG.6a are discussed below.

A modifier module may, for example, be constituted as follows: Theprimary parts of the modifier module may consist of a scaffold body 354and a modifier agent population 352.

Optionally, the module further comprises a semi-permeable membrane thatsurrounds the population of modifier agents and, in conjunction with thescaffold, encloses that population.

Semi permeable membrane 353 is a membrane across which constituents fromchamber 311 cross to react with modifier agent 352. Bellows-like cover362 surrounds the scaffold body to isolate it from contamination fromthe external environment, (see FIGS. 6a, 6c ).

The entire modifier module needs to be enclosed to protect it againstcontamination. Cover 362 is inner most protective layer that isflexible, bellows like. The bellows 362 (FIG. 6c ) is represented incollapsed form in FIG. 6a . As the modifier is inserted into reactorchamber through opening 358, covering 362 (or 370) collapses duringinsertion without exposing the modifier within. Coverings 365 and 366provided a rigid enclosure. Positioners 367 serve to position and retainthe modifier within the rigid housing 365. Body 355 is fixed by theledge 367.

Channel 358 in adapter 356 directs the modifier scaffold end 354 intothe reactor chamber through port 331 in the reactor top.

The modifier agent population 352 may be part of the scaffold body 354,directly attached to it or unattached to it but enclosed (and preferablystacked) against the scaffold body 354 with a retaining porous (fullypermeable) or semi porous (semipermeable) membrane 353.

The representation of the modifier agent populations 352 in the FIGS.6a-6d is highly schematic since the agents are so much smaller than thefilter systems, scaffolds, and other items represented in those Figures.In FIG. 6c , each agent is represented by a small hexagon. If themodifier agent is an antibody, for example, the number of hexagonsneeded to represent the agent population would be so great that thehexagons would not be recognizable. However, the representation in FIG.6c provides the clearest basis among FIGS. 6a-6d for discussing thegeometric relationship of the agents to the scaffold and anysemi-permeable membrane surrounding the agents.

In FIG. 6c , the agent population 352 consists of a two sub-populations:a sub-population of agents touching the scaffold 354 and asub-population of agents touching the semi-permeable membrane 353. Assuch it can be considered to be a schematic representation of a stackedagent population retained within the compartment by the membrane andstacked against the scaffold 354.

In FIG. 6c , Elimination of the sub-population of agents that touchesthe semi-permeable membrane 353 would provide a schematic representationof the embodiment where the agent population is bound to the scaffold.In that case, the semi-permeable membrane 353 optionally may beremoved—the decision being based on part on whether a filtering by sucha membrane is desired.

FIGS. 6a, 6b and 6d , provide in schematic fashion a representation of apopulation of modifier agents bound to the scaffold.

Part of the scaffold 354 may function as a scaffold structural “head”355. As illustrated in FIGS. 6b and 6c , the scaffold head can serve asa place of attachment for membrane 362. The scaffold head can, as shownin FIG. 6c , have a greater diameter at the top of the scaffold than atthe lower portion of the scaffold, head, thereby providing a lip thatcan sit on the module support ring 367 which ring is attached to thehousing 365 of storage case 371, a case adapted for storing a sterilizedmodifier module until the module is needed.

In FIG. 6c , the storage case 371 comprises additional features designedto hold the modifier module in place: A lower adapter 356 whose positionwithin housing 365 is fixed by the circular holder ring 368 attached toboth the holder 356 and the housing 365 of storage case 371.

The objective of membrane 353 is to retain the modifier agent population352 against the scaffold and allow fluid exchange across its wall.Therefore, If module 351 shown in FIG. 6c is inserted into chamber 311of system 301 in FIG. 6a as the module 351 in that Figure, fluid inreactor chamber 311 will be free to flow across porous membrane 353, andto contact the modifier agent population 352; thereby, allowconstituents in the reactor chamber fluid to react with the modifieragent population. The modifier agent population may comprise any agentscapable of reacting or interacting with selected constituents in thereactor chamber fluid. For example, the modifier agent may be anantibody, in which case it would be chosen to be one reactive againstsome component in the reactor chamber fluid. Alternatively, however, itmay be more effective to link the agent, such as an antibody, to somesolid resin. The resin in that case may be directly linked to, or coatedonto, the scaffold 354. The screen pore size would be smaller than theresin to retain the resin and allow free exchange across its surface.Both ends of the screen or membrane 353 would be sealed against thescaffold body so as to fully enclose and retain the resin.

Filtrate generated during the process may be allowed to flow freely orin a selective manner through pores 318, in barrier 319, into thereactor chamber 311, immersing the modifier module 350. If we assumethat an attached antibody on the resin is against a secreted agentproduced by cultured cells, then the agent will be captured by theantibody and selectively removed from the reactor chamber fluid. Thesystem 301 illustrated in FIG. 6a has many uses, one of which isdialysis. A catheter provides, as a result of its insertion in aperson's vein, a direct or indirect fluid connector 333 between thesystem and the person. In this example, the reactor chamber is providedwith a defined dialysate solution. Such a solution may be customized toprotect the user, to assist in the dialysis process, to enhance theefficacy of the modifier or effect the process in some other way. Theretentate chamber 345 of the filter element (e.g., the fibers of a HFfilter cartridge) in this case may be a micro filter membrane or ultrafilter membrane. Barrier (wall) 319 may also be selective in nature tofacilitate further fractionation of blood constituents such that onlythe desired molecules escape into the reactor chamber to react with themodifier. Blood will cycle reversibly between the alternating pump 304and the venous system, through the filter element 305. As previouslydescribed, during the alternating tangential flow filtration cycle,fluid not only flows through the lumens of the hollow fibers but it alsofacilitates bidirectional fluid flux across the filtration membrane 317between the retentate and filtrate chambers, across barrier 319 betweenthe filtrate and reactor chambers 310 and 311, respectively, and acrossbarrier 353 or other barriers that are part of the modifier module, inboth directions; the alternating flow therefore enhances mixing betweensystem compartments. Using hollow fibers with the appropriate MWCO(molecular weight cutoff) may be used for selective retention ofessential constituent of blood, cellular fraction, proteins, etc. Alsodepending on the MWCO of the membrane, other, selective, molecularconstituents may be allowed to exchange across the membrane/selectivelypermeable barrier 317 surface. It becomes possible to allow urea,certain proteins, hormones, toxins, byproducts, etc, to exchange acrossfilter membrane 317. Selective membrane 319 and potentially selectivemembrane 353 may be used to further regulate the flow of specifiedconstituents from the filtrate chamber 310 to the reactor chamber 311.The selectivity of membrane/barrier 319 may be established bycontrolling its charge properties, permeability, porosity, chemicalnature, etc. The configuration of barrier 319 may be modified toincrease its surface area.

Once in the reactor chamber 311, the product of interest can freelyexchange across barrier 353 to react or bind with the modifier agentpopulation 352. Once bound, the selected product is prevented fromreturning into the retentate or blood stream. Another possibility is touse an enzyme(s) in place of an antibody as the modifying agent. Theenzyme would be selective for a particular harmful component incirculation or to affect some critical metabolic reaction beneficial tothe patient. If that component flows from the circulation across theselective membrane(s) into the reactor chamber, it would be available toreact with the attached enzyme(s) modifier. The altered component,either inactivated or made more potent, will be free to exchange acrossbarriers 353, 319 and 317 to reenter the retentate or blood stream or toreact with a second modifier module (not shown) in the reactor chamber,for further modification or removal of fluid. In a similar manner, onecan foresee many other uses for the described configuration of theinvention, in the healthcare or other fields.

It is also foreseen that a sterile system may be supplied separatelywith a presterilized modifier module(s) 350 and 351. One has the choicefrom various modifier modules to further increase the flexibility of thesystem. It becomes possible to insert a selective modifier module 351into the reactor chamber 311 as the need may arise or based on immediaterequirements. This modular concept, in addition to providing thecapability to select from a variety of potential modules 351, alsoprovides the ability to use modifier modules 351 containing a labilemodifier agent population 352; such as proteins, that cannot be normallysterilized preassembled with the system, but can be sterilized orsanitized by other less severe means; for example, assembly of themodifier module 351 separately may involve using steam, radiation, etcfor the nonlabile parts of module 351, using filtration to sterilize thelabile components and assembly of the two in a sterile environment. Theuse of antibiotics or other preservatives offer other sanitizationoptions. Or one may store labile modifier agents in a stable form suchas frozen or freeze dried, then prior to use the modifier agentpopulation may be hydrated and activated.

It is possible that the insertion of the modifier module into anenclosed bioreactor system of the present inventions may have to takeplace in the “field” in the open, in an unprotected, unsterile,environment, a procedure that has the potential to result incontamination the system. The enclosed bioreactor system described offerthe capability of performing the procedure in an aseptic manner. Suchconnectors are commercially available the clean-pack (from Pall, Inc)and the DAC (from GE); such that, anyone familiar with the use of suchconnectors may effectively insert the modifier module into the enclosedreactor system in a sterile manner.

Product Concentration Device, System and Process

There is a growing list of products being produced by cell culture; theproducts may include proteins which may be natural or recombinant,cells, or any product that may be produced by the cellular syntheticmachinery. The production cultures may be mammalian cells, insect cells,plant cells, yeast, bacteria etc. . . . . Considerable effort has goneinto improving methods for producing such cellular products; theseinclude improvements in the methods of growing cells, including,manipulating the cells to express product, improving the ability tosustain cultures in production phase at very high cell concentrations,improving stability of the process as well as other improvements. Todaythere are cultures that can be grown to very high cell concentrations,>100×10⁶ cells/ml (greater than 100 million cells/ml) which generateproduct at ever increasing concentrations. A key bottle-neck thatresults from such improvements is in the ability to harvest the productefficiently without damaging the product or the culture. Currently,product harvesting is achieved in a number of ways. The following aresome examples of product harvesting: (1) In cases where the productionculture is taken to its termination, (e.g. batch, fed-batch, limitedperfusion), and where the product is soluble in the culture medium, theentire culture is subjected to a fractionation process that separatescells from media plus product. The separation process is typicallyachieved by centrifugation or by filtration. (2) In a continuous cultureprocess, such as “perfusion”, the product may be separated from thecultured cells continuously. The separation process may involvefiltration, cell settling or centrifugation. The product is removed fromthe culture and processed appropriately, while the cells are retained inthe culture vessel. (3) Continuous fed batch is a process, demonstratedby Kearns (U.S. Pat. No. 5,286,646), that uses a filter with pore sizecapable of retaining the cells and product in the culture, whileallowing harvesting of material smaller than the pore size of thefilter. Kearns demonstrated that removal of the lower molecular weightfiltrate supports continued cell growth and increased product formation.

In examples (1) and (3) of product harvesting, the cells and product aremaintained in the culture until its termination. Approaching itstermination, the culture typically loses viability and cell integrity isunable to be maintained; it is also a period when increasing number ofcells rupture and release their content into the culture. The viabilityand integrity of the cells typically begin to deteriorate significantlyprior to the end of the culture. The product may, therefore, besubjected to the toxic environment of the culture, subjected todigestive enzymes, shear, temperatures and other conditions not ideal tothe stability of the product. Other effects, particularly in continuousfed batch (example (3)), where cells and product may be at very highcell concentration, is the potential for formation of multimers,modification of protein glycosylation, as well as other potentialfactors that may modify the product. The effects of such adverseconditions on the product will vary from product to product. Someproducts are highly unstable and rapidly deteriorate in culture; otherproducts are more stable and can tolerate such adverse condition forlonger periods.

In example (2) of product harvesting, perfusion, the product can berapidly removed from the toxic environment of the culture and stored inan environment that would preserve its stability, including storage atreduced temperatures, pH stabilized media, etc. Two primarydisadvantages of the perfusion are: one, lower product concentration inthe harvest, as the product is not allowed to accumulate in the culture;two, large volumes of harvest that requires storage and processing torecover the product. It is preferable, therefore, to take advantage ofthe process which removes the product continuously from the toxicenvironment of the culture, yet provide the product continuously inconcentrated form at reduced volumes. Such a system is described herein.

FIG. 8 shows a system with a continuous two stage system, Unit 1 andUnit 2. In the first stage, Unit 1, the product is continuously removedfrom a culture, while retaining the cells in the culture. In the secondstage, Unit 2, the product stream from Unit 1 is concentrated by afiltration device, preferably an alternating tangential filtrationdevice, capable of retaining and separating the product from lowermolecular weight constituents, primarily water in the product stream.

In FIG. 8, a culture vessel or bioreactor system 30, analogous to vessel2 shown in FIG. 1a , is connected to input line 3 of separation device 1(also referred to as Unit 1).

While the cell separation device shown in Unit 1, FIG. 8, is afiltration device based on an alternating tangential flow filtrationsystem (described earlier and in U.S. Pat. No. 6,544,424), it is notlimited to that particular system or to filtration. Unit 1 mayconstitute any cell separation device capable of retaining cells inreactor 30 while permitting the product and waste media to be removedfrom the culture; such devices may include ones that operate based oncell settling, centrifugation, electric or magnetic fields as well asother techniques. Therefore, while all such cell retention devicesretain the cells in vessel 30, they also generate a cell depleted steam,(henceforth, also labeled “product stream” or “filtrate stream”), thatcontains the product of interest as well as the waste generated by theculture.

Although not limited to any particular cell retention or cell separationsystem, the alternating tangential flow system shown in FIG. 1a and asUnit 1 in FIG. 8 is used to illustrate the process of the invention. InUnit 1, a filter element is used with pore size that retains the cellsand allow most biomolecules including the product (e.g., IgG of 150 kDa)and waste media to be removed in the filtrate stream. A filter elementconsisting of a membrane with 0.2 micron pore size may be such a filter.In FIG. 8, the filtrate product stream emerging from the filter into thefiltrate chamber 17 of filter element 5 may be further directed throughchannels in filter housing 19 of filter element 5 and collected inchamber 11. The filtrate stream can be removed directly from thefiltrate chamber 17 within filter element 5 or from chamber 11. In thelatter case, the filter stream is removed through port 12 into tubing 13which directs the filtrate stream to Unit 2 reservoir 60 via port 66. Apump 14 controls the rate and direction of fluid transfer betweenseparation Unit 1 and separation Unit 2. Pump 14 therefore serves as aone-way valve, preventing back flow of fluid from Unit 2 to Unit 1;alternatively, a non-pump one way check valve, (not shown}, may replacepump 14 in line 13 to prevent such back flow.

Unit 2 may be any filtration system, including one based on dead endfiltration, or tangential flow filtration, but preferably one based onalternating tangential flow filtration. As illustrated in FIG. 8, likethe previously described alternating flow filtration system of Unit 1,Unit 2 contains an alternating tangential pump 84 capable of generatingan alternating tangential flow between the pump liquid chamber 77 andreservoir 60 through filter element 75. As before, controller 78, usingconduit 83, alternately pressurizes or depressurizes chamber 88 of thepump relative to reservoir 60. A flexible diaphragm, bladder or aircolumn 86, allows the transfer of pressures in chamber 88 of the pump tochamber 77. The resulting alternating pressures changes in chamber 77relative to reservoir 60, is used to generate and control alternatingtangential flow or reversible flow between chamber 77 and reservoir 60through a filter element 75.

Reservoir 60, in addition to providing a storage container formicro-filtrate 69 from Unit 1, also serves as a reservoir for the fluid69 flowing reversibly from and to pump chamber 77. Fluid reservoir 60can serve as a single liquid reservoir or interconnected with two ormore liquid reservoirs (not shown) through connecting conduits (notshown) which allows fluid flow between such reservoirs. The reservoirsmay serve as temporary storage buffer for filtrate generated in Unit 1or for modifying such filtrate prior to addition to reservoir 60;whether singly or in multiples the term reservoir 60 is applied.Additionally and optionally, the content in fluid reservoir 60 may bemonitored by probes to determine fluid level 61 within the reservoir, todetermine its pH, temperature, turbidity, for spectroscopic analysis orconductivity as well as other parameter; such information may be used toadjust indicated parameters to the desired settings by known methods.Conditioning the fluid in reservoir 60 may be used to stabilize thecontent in the reservoir or to facilitate separation of desiredconstituents. A device such as a manifold invention described in thepresent application (FIGS. 5a and 5b ) connected to reservoir 60 throughline 79 may be used to monitor the conditions in reservoir 60 and toaffect such conditions. Reservoir 60 while passive in nature, serving asa container for filtrate flowing from Unit 1, one can also envision amore active system, where reservoir 60 consists of a second alternatingtangential flow pump, where fluid flowing from pump 84 is received insuch second pump liquid chamber; conversely, the second pump may bepressurized to cause fluid in its liquid chamber to through the filtermodule 75 to pump 84 liquid chamber 77. Such reversible and alternatingflow may be used in a manner that would maintain the filtration processin Unit 2 under continuous positive pressure, which may be beneficial inmany applications. Furthermore, in yet another variation a knowledgeableuser may invert the process described above, where reservoir 60 is shownin the preferred form and its preferred use; for example, itsconceivable that chamber 81 in Unit 2 may serve as a container formicro-filtrate 69 emanating from Unit 1, while reservoir 60 may serve asan ultra-filtrate reservoir; in which case, the filtration directionthough filter element 75 of Unit 2 will be conducted in reverse.(Reservoir 60 need not be attached to housing 2, a conduit from theentrance end of the filter element 75 to reservoir 60 is also possible.)Fluid 69 would be concentrated in chamber 81 and externally to thehollow fiber in chamber 17, while low molecular stream filtered throughthe ultra-filter will flow into the lumen of the hollow fibers and intoreservoir 60 from where it can be removed through line 73. Theconcentrate can be removed from chamber 81 through line 64, whichextends into chamber 81 through tubing 62.

Hollow fiber module 75 within the housing 2 of Unit 2 may be selectedfor desired properties, specifically for its pore sized; for example, apore size may be selected that will retain constituents, such as theproduct, larger than the pore size; while constituents smaller than thefilter membrane pore size and water will pass thorough the membrane asultra-filtrate; for example, an ultrafiltration membrane with a 50 Kdapore size is commonly used to separate proteins such IgG antibodies fromsmaller MW constituents. Therefore, micro-filtrate or product stream 69within reservoir 60 flowing reversibly between said reservoir and pumpchamber 77 through such a selective hollow fiber module can be used asan efficient filtration process to retain the product but allowparticles smaller than the filter pores, particularly water to beremoved from the product stream 69. The resulting ultra-filtrate or“waste stream” can be collected in the filtrate chamber 87 of the filterelement 75. The filter element 75 may provide openings 89 in itsenclosure for the filtrate to flow from the inside of module into afiltrate collection chamber 81 enclosed peripherally by the Unit 2containment wall 85. From chamber 81 the filtrate may be harvestedthrough port 62 and line 63 and pump 64. Alternately the filtrate may becollected directly from the filtrate chamber 87 bypassing chamber 81.The rate of ultra-filtrate collection through port 62 and line 63 may becontrolled with harvest pump 74. Removal of ultra-filtrate andparticularly water from the micro-filtrate product pool 69 willconcentrate the product within reservoir 60. (The product can beconcentrated two fold, preferably it may be concentrated four fold, morepreferable still, the product may be concentrated more than ten fold,The volume of suspending medium will be decreased inversely to theconcentration). The rate of concentration of the product will bedetermined not only by the rate of water removal from the micro-filtrateproduct pool 69 but also by the following factors: Product concentrationwill be effected by the rate of micro-filtrate flow from Unit 1 intopool 69, diluting the product in the pool. Product concentration in pool69 will also be effected by the rate of concentrate removal from pool69; such removal may be achieved through port 76, line 73 and pump 74.Product concentration in pool 69 may also be effected by optionaladdition of fluids from an external source, as needed to condition theconcentrate to facilitate its further processing. In summary, theprocess provides the user the means for continuous concentration of theproduct, the means for controlling product concentration, including therate of product concentration and the rate of product harvest in itsconcentrated form.

The system offers the ability to maintain a culture in perfusion toachieve high cell concentrations and high product through-put. It offersthe ability to remove the product from the toxic environment of theculture in a vessel 30 connected to line 3. Optionally it offers theability to condition the product stream produced by Unit 1. It offersthe ability for continuous concentration of the product in Unit 2 andprovides a continuous stream of concentrated product. It provides themeans for controlling the indicated flows. Unit 2, in part or fully, maybe maintained at lower temperature to preserve the product;additionally, the emanating concentrated product stream may also bepreserved at lower temperature for subsequent processing or forimmediate modification as required for purification or other procedures.

In a general aspect, the product concentration comprises:

-   -   1) a hollow fiber filter element (or module; preferably a        cylindrical hollow fiber filter cartridge) said filter element        comprising an entrance end and an exit end, said filter element        further comprising a plurality (more than one) of filtration        retentate chambers, each filtration retentate chamber being an        open-ended hollow fiber, wherein each fiber comprises a        semi-permeable outer wall, said filter element further        comprising a filtrate chamber, said filter chamber comprising a        filtrate chamber inner wall and a filtrate chamber outer wall,        such that said filtrate chamber encloses said fibers but does        not block their open ends, such that the semi-permeable outer        walls of the fibers are also part of the filtrate chamber inner        wall, said filtrate chamber outer wall optionally comprising        pores or outlet(s) for flowing the filtrate from the filtrate        chamber to an intermediate or final collection vessel;    -   2) an alternating flow pump, said pump said pump connected to        the exit end of the filter element so as to permit fluid from        the pump to enter the filtration retentate chambers and fluid        from the retentate chambers to enter the pump (said pump, for        example, comprising a pump housing, two chambers, and a        diaphragm separating the chambers);    -   3) A reservoir connected to said filter element entrance end, so        that fluid can flow between said reservoir and the retentate        filtration chambers;

wherein the retentate chamber fibers are disposed in parallel to thecenter axis of the filter element (the center axis extends through thecenter of the cylinder from one filter element end to the other, whereinthose fibers each have an entrance at the entrance end of the filterelement and an exit at the exit end of the filter element. (In oneembodiment, the pump does not comprise an open drainage tube that wouldallow removal of retentate from the filtration retentate chambers).

In the case where the filtrate chamber outer wall contains pores, theproduction concentration device further comprises a filtrate collectionchamber, said filtrate collection chamber disposed so that it at leastpartially encloses both the filtrate chamber and the filtrationretentate chamber in a sealed manner but does not block fluid flow inand out of the filter element entrance, said filtrate collection chambercomprising a filtrate collection chamber inner wall and a filtratecollection chamber outer wall, said filtrate collection chamber innerwall comprising the filtrate chamber outer wall;

In the case where the filtrate chamber wall contains pores, theproduction concentration device may further comprise a filtratecollection chamber harvest line and/or a reservoir adapter line, saidfiltrate collection chamber harvest line connected to the filtratecollection chamber so as to allow fluid to be harvested from thefiltrate collection chamber, said reservoir adapter line connected tothe reservoir so as to allow fluid to be harvested from the reservoiradapter.

In a particular aspect, the product retention device further comprises aline (tube) connected to the reservoir so that fluid can enter thereservoir from an external source of fluid; wherein a one-way valveand/or pump connected to or in said line so that fluid can flow from theexternal source into the reservoir but preferably not in the reversedirection.

In another general aspect of the invention, a product retention systemrelated to the product retention device for modification orconcentration of a cell-depleted filtrate, comprises:

1) a product concentration device as specified in claim 1 or 2;

2) a cell depletion device, said cell depletion device capable ofgenerating a cell-depleted product fluid, said cell depletion devicecomprising a chamber where the cell-depleted fluid resides, said chamberconnected to the line (tube) of the product concentration device.

In a particular aspect of the product retention system, the chamber isconnected to the line of the product concentration device.

In a particular aspect of the product retention system the celldepletion device has a component selected from the group consisting of afilter, a centrifuge or other cell separation devices.

In a particular aspect of the product retention system, the celldepletion device comprises a filter, said filter capable of preventingthe passage of cells while allowing the passage of product in fluid.

In a particular aspect of the product retention system, cell depletiondevice comprises a centrifuge, said centrifuge capable removing cellsfrom a portion of a fluid comprising product, thereby creating acell-depleted product fluid.

In a related process, in a general aspect, the process comprisesutilizing an aforementioned product concentration device such the resultof the process is to generate a product concentration greater than theproduct concentration in the cell depleted fluid at the start of theprocess.

What is claimed is:
 1. An enclosed filtration system process, saidprocess comprising the steps of: 1) discharging fluid from a retentatechamber via a fluid connector into a vessel such that during saiddischarging a portion of said fluid is directed via a semipermeablebarrier into a filtrate chamber and is then directed via a selectivebarrier into a reactor chamber, wherein said discharging is due to theforce exerted by a diaphragm pump connected to the retentate chamber ata position distal to the fluid connector; and 2) reversing the directionof the force exerted by the diaphragm pump so that at least some fluidfrom the vessel flows back into the retentate chamber, at least somefluid from the retentate chamber flows into the filtrate chamber andsome fluid from the filtrate chamber flows into the reactor chamber; and3) repeating steps (1) and (2) at least once, wherein fluid dischargedfrom the retentate chamber is selected from the group consisting of asuspension and a solution, and wherein the retentate chamber, filtratechamber, reactor chamber, and diaphragm pump together form an enclosedfiltration system, wherein (a) said retentate chamber of said enclosedfiltration system comprises an entrance at its entrance end and an exitat its exit end, said retentate chamber comprising a retentate chamberwall, at least a portion of said wall being semi-permeable; (b) saidfiltrate chamber of said enclosed filtration system at least partiallyencloses said retentate chamber, said filtrate chamber comprising afilter chamber inner wall and a filter chamber outer wall, wherein atleast a portion of the filter chamber inner wall corresponds to thesemi-permeable portion of the retentate chamber wall; said filtratechamber outer wall comprising a filtrate chamber outer barrier; (c) saidreactor chamber of said enclosed filtration system is disposed so thatit at least partially encloses both the filtrate chamber and theretentate chamber in a sealed manner but does not block fluid flow inand out of the retentate chamber entrance, said reactor chambercomprising a reactor chamber inner wall and a reactor chamber outerwall, said reactor chamber inner wall comprising the filtrate chamberouter barrier, said reactor chamber outer wall being sealed to theoutside of either the retentate chamber or to the outside of thefiltrate chamber, said reactor chamber outer wall optionally sealed tothe alternating pump outer wall; and (d) wherein said enclosedfiltration system further comprises (i) an alternating flow pumpconnected to the perimeter of the retentate chamber exit so as to permitfluid from the pump to enter the retentate chamber and fluid from theretentate chamber to flow into the pump; said pump comprising an outerwall, a diaphragm, and two chambers separated by the diaphragm, and (ii)a harvest port attached to the reactor chamber outer wall so as to allowfluid to leave or enter the reactor chamber.
 2. The process of claim 1,wherein said enclosed filtration system is presterilized.
 3. The processof claim 1, further comprising harvesting at least some of the fluidfrom the filtrate chamber.
 4. The process of claim 1, further comprisingmeasuring a culture condition using at least one probe, the at least oneprobe enclosed within said enclosed filtration system.
 5. The process ofclaim 1, further comprising a processing chamber connected via a fluidconnector to said retentate chamber.
 6. The process of claim 5, whereinthe processing chamber is single-use.
 7. The process of claim 5, whereinthe processing chamber comprises one or more ports in a processingchamber wall.
 8. The process of claim 7, further comprising adding fluidthrough one of the one or more ports in the processing chamber wall. 9.The process of claim 5, wherein the processing chamber is a bag.